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Picking up Plasmid DNA using Nanodrop, but not using Electrophoresis

Picking up Plasmid DNA using Nanodrop, but not using Electrophoresis


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I am currently trying to see if 2 bacteria contains plasmids or not. I had used promega plasmid extraction kit on the bacteria. I ran a gel electrophoresis (.8% gel) with the extract product, however its not showing the presents of any DNA. I also ran the product through Nanodrop (2000) and these where the concentrations I got:

1 : 6.5 ng/ul & 260/280 = 1.56
2 : 49.4 ng/ul & 260/280: 1.96

Based on experience, I want to say #1 contains no plasmids, however #2 may have a small concentration of plasmids. Please give me your opinion on what to conclude. Also, if there is another test I can try to do to confirm plasmid presents or not, please let me know.


I don't know how much culture volume you used, but the amounts you have look more like a medium/high copy plasmid. I don't know if that's what you'd expect for a natural plasmid.

In addition to the other answer, it could also be sheared genomic DNA, cosmid (that would run too high on your gel to see), or maybe just nucleotides (if you messed up the prep in some way).

Another strategy to figure out if your strain has plasmids depends on what else you know about these strains. If you don't know that much: do 16S sequencing, it might be a known strain with known plasmids. If you already know it's a newly discovered strain: sequence the whole thing. Even with relatively low coverage the presence of plasmids will be quite clear. It's a bit more expensive than a plasmid prep + gel, but you'll also get a lot of other information about the organism.


Having a positive reading on the Nanodrop but no band on the gel can be due to a number of different issues (not exhaustive):

  1. An incorrectly zeroed sample
  2. Incomplete staining of the gel
  3. Contamination of the sample
  4. Human errors in loading of samples

You should try to exclude these possibilities by positive controls in order to find out the true reason behind the inconsistent results.


I would recommend switching up your PCR cloning program. Often, even after purifying DNA and RNA in conjunction with great care PCR can be the bottle neck for Gel Electrophoresis, as well as ensuring the quality (depth, void of blunt edges, etc) of the agarose gel- 49 ng/ul is not enough for progression. If all that still doesn't improve your case then it may be an issue with cultivating bacterica cultures -after purifying plasmids from bacterial clones vials should appear very cloudy (8-16 hrs was my go to time period), futhermore make sure to avoid light exposure on to the agarose gel as well as keeping it clean and submerged under cold PBS before inserting your buffered DNA (I used 10 microliters - but it tends to vary depending on the thickness of your gel) Also, when I would use a higher voltage during gel EP I would have a higher chance of having vertical streaking though it may be faster.

If nothing works… then I can only allude to possible human errors. Last thing! I hope you clean the nanodrop before you take count, having protein in your product can be a result of that. good luck! bacterica need love too!


Depending on how you followed the protocol you could theoretical also have bacterial genomic DNA in your sample. If you follow the protocol strictly this should not happen, but if so, you would be able to measure the genomic DNA. In the agarose gel, however, it would usually be stuck around the top since it is too large to migrate.

Vortexing the sample before loading the column could cause this, for example (see Promega PureYield Manual).


20 Funny DNA Jokes And Pick Up Lines… With Explanations

From unzipping genes to DNA contamination, here are some funny DNA jokes and pick up lines.

Here are some DNA jokes for all of us molecular biology and science nerds out there. Explanations are included for the rest of us. Enjoy!

Bill Clinton&rsquos DNA

The test on the dress came back inconclusive. Everyone in Arkansas has the same DNA.

Explanation: Monica Lewinsky and Bill Clinton had a secret affair while he was President Of The United States. After an encounter with Clinton, Lewinsky saved a blue dress that had Clinton&rsquos &ldquoDNA&rdquo on it. Clinton is from Arkansas. Sometimes people joke about people in the south all being related.

No Genes

Explanation: Many high-end nightclubs don&rsquot admit patrons wearing jeans. Jeans and genes are homonyms.

DNA Ligase

DNA Test Results May Vary

Explanation: Many people who have tried DNA testing services like 23andMe and AncestryDNA have gotten surprising results.

Stop Copying Me

Pick Up Line: Falling in love with you takes less time than my DNA takes to replicate.

DNA Evidence

Explanation: DNA testing and evidence have had a dramatic impact on the rate of both current and cold case closures. As a result, criminals now have a much harder time getting away with crimes.

What Does DNA stand For?

DNA Contamination

Explanation: One of the biggest challenges for forensics investigators and scientists is avoiding DNA contamination.

DNA-Spaghetti

Rearrange The DNA

Pick Up Line: DNA spelled backward is AND, like&hellip me AND you.

Unzip Your Genes

Pick Up Line: Baby, I wish I were DNA Helicase, so I could unzip your genes.

Explanation: A DNA helicase is an enzyme that can &ldquounzip&rdquo a strand of DNA down the middle of the helix.

DNA Helicase And Perverts

  • Q: What&rsquos do DNA helicase and perverts have in common?
  • A: They both want to unzip your genes

Explanation: A strand of DNA can be &ldquounzipped&rdquo with a DNA helicase enzyme.

DNA And Diarrhea

DNA Fat Joke

Explanation: The words jeans and genes are homonyms.

Restriction Enzyme

Am I Adopted?

Fred came home from University in tears.

&ldquoMom, am I adopted?&rdquo

&ldquoNo of course not&rdquo, replied his mother. Why would you think such a thing?

Fred showed her his genealogy DNA test results. No match for any of his relatives, and strong matches for a family who lived the other side of the city.

Upset, his mother called her husband. &ldquoHoney, Fred has done a DNA test, and&hellip and&hellip I don&rsquot know how to say this&hellip he may not be our son.&rdquo

&ldquoWell, obviously!&rdquo he replied.

&ldquoWhat do you mean?&rdquo

&ldquoIt was your idea in the first place&rdquo her husband continued. &ldquoYou remember, that first night in the hospital when the baby did nothing but scream and cry and scream and cry. On and on. And you asked me to change him.&rdquo

&ldquoI picked a good one I reckon. Ever so proud of Fred.&rdquo

DNA Printer: DNA Test Jokes

Explanation: This DNA test joke shows two strips of paper coming out of the printer forming a helix, the same shape of DNA.

Pasta DNA

Explanation: The spiral shape of this pasta noodle looks similar to a DNA helix.

The Petting Zoo

Q: What do you get when you mix human and goat DNA?

A: Kicked out of the petting zoo.

Explanation: Sorry we&rsquore not explaining this one.

NERD NOTE : Have you ever wondered: &ldquoWhat does DNA stand for?&rdquo DNA is the shorter abbreviation for deoxyribonucleic acid. Your DNA consists of two different nucleic acids. Deoxyribonucleic acid (DNA) is the main acid. The secondary acid is ribonucleic acid (RNA).


Results

Universal MCS and its associated primer design

The design of the UMCS for universal subcloning is shown in Fig. 2a. The main features of this novel MCS design are as follows: (1) The length of UMCS is only 48 bp, which is shorter than many of the existing MCSs. The actual sequence to be synthesized is 42 bp for both the forward and reverse directions (Fig. 2b). By annealing and ligating to a predigested vector molecule, the plasmid with the UMCS was reconstituted. (2) The restriction enzyme recognition sites at both termini are needed only for UMCS integration. They can be replaced freely according to the original MCS compatibility of the vector. (3) The restriction enzyme recognition site in the middle of the UMCS is used only for vector linearization, which can be substituted with any other sequences for digestion if they are compatible with the vector. (4) The two homologous linker regions were carefully optimized to have close Tm and the same GC content, which are supposed to facilitate PCR. Hairpin and dimerization of these regions were also minimized through optimization studies. (5) Primers used for the amplification of the insert can be easily designed by introducing a homologous linker at their 5′-terminus, while the Tm of the template binding region is calculated to be approximately 55 °C (Fig. 2c). (6) Theoretically, because of MCS consistency, any vector with a UMCS sequence can be amplified by the “UMCS-PCR-F + UMCS-PCR-R” primer pair, and any insert cloned into the UMCS can be amplified by the “UMCS-Seq-F + UMCS-Seq-R” primer pair. (7) Both linker regions do not contain rare codons [23, 24]. If the linker sequence is to be expressed, the codons contained in the UMCS are unlikely to affect the target protein expression. This is crucial for vectors that express N-terminal tags.

Principle of the UMCS and corresponding primer design. a The UMCS sequence used in this study contains two homologous linker regions that are connected by the SalI recognition sequence. The flanking HindIII and XbaI recognition sequences were designed for replacing the original MCS. All three recognition sites can be freely substituted for other sequences. b The UMCS was first synthesized as two ssDNAs (each with a length of 42 bp). After annealing and ligating, the UMCS was incorporated into the vector, thus replacing the original MCS. c The recommended primer design to maximize assembly efficiency. The homologous linker region (12

15 bp) is included in the 5′-terminus of each primer, and the Tm of the template binding region was designed to be approximately 55 °C

Construction of pUC19-UMCS-EGFP

To demonstrate proof of principle, we first constructed two types of pUC19-UMCS-EGFP vectors (Fig. 3) as tools for studying the transformation efficiency. These vectors are modified versions of the pUC19 because the lacZ sequence was first replaced by UMCS-XhoI sequences with different linearization sites (SalI or EcoRV, as shown in Fig. 3a and 3b), followed by the insertion of EGFP (Enhanced Green Fluorescent Protein) through XhoI-based digestion and ligation. Among them, SalI represented the linearized product with sticky ends, while EcoRV represented blunt ends. Together with vectors linearized by PCR, the effect of different linearization methods on transformation can be clarified. During in vivo assembly, if the mCherry CDS is successfully cloned into the UMCS, the colony will appear orange. Otherwise, it will be green or colorless (through nonspecific assembly). By calculating orange colonies against the total number of colonies, the positive ratio of transformation can be determined.

Schematic depicting of pUC19-based plasmid models. Two different designs of the pUC19-UMCS-EGFP plasmid with SalI or EcoRV recognition sequence were used for sticky- or blunt-end digestion

Effects of the linearization method and homologous linker length

Next, we evaluated the effects of different linearization methods and the length of the homologous linker in terms of the number of transformants and the corresponding positive ratio. As shown in Fig. 4a, when PCR was used as a linearization method, the transformation efficiency showed a positive correlation with the length of the linker sequence. When a 6-bp linker was used, less than 100 CFU/plate and a positive ratio below 10% were confirmed. In contrast, the efficiency of transformation increased to 259 ± 34, 742 ± 132, 1306 ± 123, and 1525 ± 165 CFU/plate, and the positive ratio increased dramatically to 78 ± 4.2%, 96 ± 1.5%, 95 ± 1.7%, and 97 ± 1.9% when 9-, 12-, 15-, and 18-bp linkers were tested, respectively. It is evident that a 6-bp linker is not enough for bacterial in vivo assembly, and a length longer than 9 bp is necessary when performing such experiments. Moreover, there were no significant differences between the 15- and 18-bp linker. Considering both the colony number and positive ratio, we concluded that the length of the homologous linker for the PCR method was 12

15 bp. On the other hand, when linearization was carried out by restriction digestion, it was shown (Fig. 4b and 4c) that the digested vectors with blunt ends (EcoRV) yielded much more positive clones than sticky ends at any of the linker lengths we tested. According to the data collected, when a restriction enzyme was used for linearization, a 12

15-bp linker also ensured enough transformants and positive clones to screen. In these cases, further increase the linker length is not necessary.

Effects of the linearization method and homologous length on assembly efficiency. a Transformation efficiency (shown as CFU/plate and positive ratio of colonies) of PCR-linearized pUC19-UMCS-S-EGFP with the mCherry sequence flanked by 6-, 9-, 12-, 15-, and 18-bp homologous linkers (n = 4). b and c Transformation efficiency of SalI- and EcoRV-digested pUC19-UMCS-S-EGFP and pUC19-UMCS-E-EGFP with the mCherry sequence flanked by 9-, 12-, 15-, and 18-bp homologous linkers (n = 4)

Effects of insert/vector ratio on transformation

The molar ratio of insert/vector is considered a critical factor for high-efficiency transformation [25]. Thus, we further investigated the influence of PCR, SalI and EcoRV digestion on transformation efficiency at multiple insert/vector levels. The yields of transformants and the positive ratio at the linker length of 15 bp were determined (Fig. 5 and Supplementary Figure S1) with molar ratios of 1, 5, 10, and 15. According to the data shown, regardless of the linearization method used, the optimal molar ratio remained at 5. A further increase in insert usage will not improve the results, while a decreased insert quantity shows a negative effect on transformation efficiency. Therefore, we used a molar ratio of 5 in all subsequent studies, which is consistent with the finding of Kostylev et al. [26]

Effects of insert/vector ratio on assembly efficiency. Effects of insert/vector molar ratio on the recombination of pUC19-UMCS-S-EGFP linearized by PCR (a) or SalI digestion (b) with the mCherry sequence flanked by 15-bp homologous linkers (n = 4)

Effects of the digestive linearization method on the fidelity of assembly

We were interested in the assembly fidelity of the digestive linearization method mainly based on three reasons: (1) Although high fidelity enzymes such as Q5, Phusion, or KOD have extremely low mismatch rates [27, 28] during the reaction, PCR products could still suffer from random errors introduced by DNA polymerase. Moreover, the possibility of introducing random mutations during the PCR process increases with PCR cycles and the length of vectors, and it is laborious to verify by sequencing. (2) Some vectors are difficult to amplify by PCR. For example, the GC content of the vector is too high or too low, or the vector contains too many repetitive sequences, which will affect PCR and even render the reaction impossible to continue [29,30,31]. (3) As previously mentioned, if vectors cannot be linearized by PCR or fidelity is vital for the experiment, the methods shown in Fig. 6 must be applied. Under such circumstances, only the sequences flanking the linearization site are used as homologous linkers to ensure the versatility of UMCS. However, when the flanking sequences (15 bp) were used as homologous linkers, the post-digestion residual bases then served as nonhomologous sequences. This means the residual bases might displace part of the insert and cause mutations at its 5′-terminus, 3′-terminus (confirmed during our pilot study), or both. The frequency of such events is a major concern for UMCS applications, since the UMCS is only universal when this frequency is low enough.

Assembly fidelity evaluation of vectors linearized by restriction digestion. a Schematic assembly of SalI-digested pUC19-UMCS-S-EGFP and mCherry CDS with 9-, 12-, and 15-bp homologous linkers. b The five possible plasmid products that can be observed after the transformation of vectors and inserts mentioned in (a). Among them, if the 3′-terminus of mCherry CDS was replaced by part of the SalI recognition sequence (“TCGAC”), the stop codon of the mCherry protein would then be destroyed, resulting in the expression of the mCherry-EGFP fusion protein with a characteristic yellow color that can be counted accurately. c Colonies with green, orange, and yellow colors can be observed when cells express different fluorescent proteins. Note that cells transformed with the pUC19 plasmid (colorless) were also spread onto the plate as a reference. d The orange and yellow colonies counted after the transformation of vectors and inserts mentioned in (a). Method fidelity can be calculated by one minus the number of yellow colonies divided by the number of orange colonies (n = 4). e-h The other experiments and results following the same procedure described in (a-d), except the vector was linearized by EcoRV digestion

Therefore, SalI (Fig. 6a-d) and EcoRV (Fig. 6e-h) digestion were taken as sticky- and blunt-end examples to evaluate sequence replacement between insert and residual bases after assembly. As shown in Fig. 6, when the mCherry CDS was used as an insert, five possible products could be expected: (1) if the assembly fails, the colony will appear green (only EGFP expression) (2) if the fragments assembled successfully and the mCherry CDS remains intact, the colony will appear orange (only mCherry expression) (3) if assembled successfully, but the 5′-terminus of the mCherry CDS is partially replaced, the colony will appear orange (only mCherry expression) (4) if assembled successfully, but the 3′-terminus of the mCherry CDS is partially replaced, the colony appears yellow for the loss of the mCherry stop codon (mCherry-EGFP fusion protein is expressed) and (5) if assembled successfully, while both the 5′- and 3′-terminus of the mCherry CDS are partially replaced, the colony also appears yellow (mCherry-EGFP fusion protein is expressed). For subcloning, we assume that the 5′- and 3′-terminus of the insert will be replaced at the same frequency, then the frequency can be derived by calculating the number of yellow colonies against the orange ones. Thus, Fig. 6d strongly suggests that the frequency of mutation at the 3′-terminus of the insert is low when SalI is used for vector linearization. In fact, less than 0.75% of positive clones contained sequence replacement at the 3′-terminus or at both the 5′- and 3′-terminus. This indicates that the possibility of having at least one mutated terminus is less than 1.5% when the length of the linker is 12 or 15 bp. Therefore, it will be very unlikely for anyone to pick up a mutated product by chance. Moreover, when EcoRV was used for linearization, the frequency of replacement events seemed less than that of SalI by having a statistical value of less than 0.3% on both 12- and 15-bp linkers. In conclusion, after digestion, the residual bases slightly affected the fidelity of assembly (an assumed mechanism of bacterial in vivo assembly with nonhomologous regions is given in Supplementary Fig. S2), but most of the positive colonies were still expected to contain the correct plasmid.

Construction of larger plasmid based on UMCS

To further demonstrate the versatility of the UMCS, we constructed an additional four vectors containing UMCS: (1) pET24a(+)-UMCS, the UMCS was inserted between the original BamHI and XhoI restriction sites, resulting in a plasmid size of 5312 bp (2) pACT-UMCS and pBind-UMCS, the UMCS was inserted between the original BamHI and XbaI restriction sites, the last “C” of the BamHI recognition sequence was intentionally removed to ensure the correct expression of the insert, resulting in the plasmid size of 5578 and 6372 bp (3) pCold TF DNA-UMCS, the UMCS was inserted between the original NdeI and XbaI restriction sites, resulting in a plasmid size of 5757 bp. Together with the pUC19-UMCS-S-EGFP that was constructed previously, we examined the transformation efficiency of cloning the mCherry (711 bp), RXRα (Retinoid X Receptor Alpha, 1389 bp), and p85α (Phosphoinositide 3-kinase p85α, 2175 bp) CDS into these vectors. During these experiments, each CDS plus a 15-bp homologous linker region (as shown in Fig. 3a) at both termini was amplified as insert, each transformation was repeated four times, and colony PCR was performed on a panel of 24 colonies randomly selected from each transformation, followed by agarose gel electrophoresis analysis to identify the positive colonies and the positive ratio accordingly. The corresponding results are shown in Fig. 7. In summary, (1) regardless of the linearization method we tried, the yield of transformants decreased as the size of the final plasmid increased (2) if PCR was used for vector linearization, no apparent correlations between the positive ratio and the size of the plasmid were observed, and the positive ratios were maintained at a high level (> 85%) in all experiments and (3) when SalI digestion was used, the positive ratio decreased with the increasing size of the final product. However, in our study, this ratio was always maintained at more than 1/3, which is enough for screening positive clones by PCR.

Additional versatility test for UMCS. a-e The efficiency of in vivo assembly according to 5 different UMCS-based vectors and 3 inserts was evaluated. All vector/insert combinations were repeated four times. For each transformation, 24 colonies were randomly picked for colony PCR and then subjected to agarose gel electrophoresis analysis. PCR products showing the same band as that of the insert were considered positive

Multi-fragment assembly

Similar to the single-fragment experiments, we used mCherry, RXRα, and RLuc (Renilla Luciferase) for multi-fragment assembly test. As presented in Fig. 8a, the 5′-terminus of the first fragment and the 3′-terminus of the last fragment must have homologous sequences corresponding to the UMCS. The linkers of other fragments needing to be assembled can be designed according to previous studies [6]. Since multi-fragment assembly significantly reduces the yield of transformants and positive clones (Fig. 8b), PCR as a linearization method or dephosphorylation after enzyme digestion is recommended. Meanwhile, further increasing the amount of DNA and the number of component cells might be necessary for the assembly of more than 3 insert fragments.

Multi-fragment assembly. a Schematic representation of multi-fragment assembly assessment. We used up to 3 different insert fragments for multi-fragment assembly experiments. Each insert has a 5× molar concentration of the PCR linearized vector. b Effects of the fragment count on transformation. The numbers of transformants and positive clones decreases with an increasing number of fragments. For each transformation, 24 colonies were randomly picked for colony PCR and then subjected to agarose gel electrophoresis analysis. PCR products showing the same band as that of the assembled insert(s) were considered positive


Sequencing problem and restricition enzyme digestion - (May/07/2013 )

Hi all,
I currently receive my sequencing result but i found some problems with it!
My work:
get dna sample -> amplified desired sequence by our primers -> run gel to confirm whether the product is correct or not -> if correct, go ahead with Restriction enzyme digestion (plasmid and pcr product) -> run gel -> if correct, go ahead with ligation -> transformtion -> pick colonies -> purify -> RE again (confirm whether the plasmid take up the DNA) -> sequencing

My target sequence should be around 500b.p. However, after ligation, I do the transformation and restriction enzyme digestion and then finally agarose gel electrophoresis again, the bands shown were not at 500b.p. Bands at different position (low M.W. and High M.w.) were shown.

Is there something contaminate the sample?

On the other hand, I would like to ask what is the ideal concentration and OD ratio for plasmid? Thank you!

Hi all,
I currently receive my sequencing result but i found some problems with it!
My work:
get dna sample -> amplified desired sequence by our primers -> run gel to confirm whether the product is correct or not -> if correct, go ahead with Restriction enzyme digestion (plasmid and pcr product) -> run gel -> if correct, go ahead with ligation -> transformtion -> pick colonies -> purify -> RE again (confirm whether the plasmid take up the DNA) -> sequencing

In my ligation, i tried out the 1:1 , 3:1 ratio of insert to vector.
The competent cells were prepared and used by all lab groups.

However, after transformation, we have found little (1:1) or actually no colonies (3:1) for most of the plates.
I pick up one of the only white colonies and uses it for later restriction enzyme digestion process.
The result of gel electrophoresis was not good. My target sequence should be around 500b.p. T he bands shown were not at 500b.p. Bands at different position (low M.W. and High M.w.) were shown.

Is there something wrong with our ligation and transformation technique?
On the other hand, I would like to ask what is the ideal concentration and OD ratio for plasmid? Thank you!

You can sequence directly from your PCR product. Purify the product and add each of the two primers separately. With a 500 bp fragment, you will likely get complete coverage.

The problem you have could be in many places. Are there 5' bases outside of your RE cut site on the primers? Do you purify the PCR product prior to the RE digest?

What is your plasmid? Is there any restriction site what you are using in the plasmid or insert? What are the enzymes? Some restriction enzymes have star activity, if you incubate long time that will chop the DNA.

Due to time-constraint and limited resources, I cannot send out my direct PCR product to sequencing. However, I would like to find out why my sequencing result is false. I have purified my product before the last RE. In my first RE, the gel electrophoresis shows that my PCR and Plasmid were cut correctly.

My plasmid is p-GEM-3z. I have blast the sequencing results. I have chosen the most similar human gene for analysis. I found the restriction sites used (bamh1 and ecor1) on that gene. However, the size of this insert (

1000b.p.) does not match the last gel electrophoresis result(where band was found at

The last restriction enzyme, due to some errors, it only incubated for around 2 and a half hour, originally 4 hours were needed.

I think after sequencing your insert you can figure out. If the restriction site bases are wrong you may not get your target gene after digestion. The enzyme added to the reaction may cut the enzyme present in the pGEMT vector.

Enzyme Cat# Temp Supplied NEBuffer Supplements % Activity in NEBuffer BSA SAM 1 2 3 4 BamHI * R0136 37°C NEBuffer 3 Yes No 75 100 100 100 EcoRI * R0101 37°C NEBuffer EcoRI No No 100 100 100 100
Double Digest Recommendation(s) for BamHI + EcoRI:

Note: The above recommendation is based on the experimental results. Please checkSuggested NEBuffers for Double Digestion.
* BamHI has a High Fidelity version (R3136)
EcoRI has a High Fidelity version (R3101)
High Fidelity (HF) Restriction Enzymes have been engineered for reduced star activity
and have 100% activity in buffer 4 which may simplify your double digest.

christy on Wed May 8 06:22:08 2013 said:

I think after sequencing your insert you can figure out. If the restriction site bases are wrong you may not get your target gene after digestion. The enzyme added to the reaction may cut the enzyme present in the pGEMT vector.

The control A sequence from the sequencing result is the p-GEM vector.
In the T7 promoter sequence, I blast the results and it is not similar with the pGEM vector
In my colonies, both white and colonies were found. Why would this happen??

christy on Wed May 8 06:23:46 2013 said:

Enzyme Cat# Temp Supplied NEBuffer Supplements % Activity in NEBuffer BSA SAM 1 2 3 4 BamHI * R0136 37°C NEBuffer 3 Yes No 75 100 100 100 EcoRI * R0101 37°C NEBuffer EcoRI No No 100 100 100 100
Double Digest Recommendation(s) for BamHI + EcoRI:

Thank you for your information. I initially tended to digest for 4hrs at 37 degree Celsius. However, due to some errors, it only digested for 2 hours. will this lead to the results i described? (many differ M.w. bands on my gel). Thank you

Most digestions are finished in 5-15 minutes. I never digest longer than 1 hour, often for 30 minutes. This should have zero effect on your sequencing.


Module 1 : DNA Tools and Biotechnology

Welcome to today’s lecture, we are going to talk about DNA tools and gene cloning,so in the previous lecture, you have learnt about the theoretical concepts of DNA cloning,today, you will get familiar with the various steps, which are involved in performing DNAcloning in laboratory setup, you will also see that how a gene of interest is insertedin a vector then it is selected based on the presence of an antibiotic gene.So, let us start the lab demonstration session now, hello everybody today we are going todiscuss about the workflow of molecular cloning.Molecular cloning has some basic steps which we are going to go one by one, so firstlywhat we are trying to do is introduce a foreign gene into an organism which we normally referredto as an insert, so this is my insert sample.So, what we do is we take our insert DNA, we introduce into a carrier molecule normallyknown as vector, so most commonly use vectors includes plasmids, sot in this case we areusing a plasmid DNA in which we are going to introduce a foreign gene our insert, sothere are as we may know plasmids, they occur naturally in bacteria’s and over the yearsstudies have been done, and you have seen that these plasmids also have some antibioticresistance gene.So, which confers a resistance against particular antibiotic to the bacteria, so if a bacteriais grown under that antibiotic condition, it will, it will its growth will not be affectedand it will grow perfectly normal.So, here we have a vector DNA, we have our insert, so in order to introduce my insertinto the vector, so let us begin with the experiment, we have taken a vector DNA also,we have an insert DNA.What we are going to do is we are going to treat them with restriction enzymes, restrictionenzymes are going to cut these DNA and they will create compatible ends, so that the insertand the vector they can join and form a perfectly circular structure.So, we are going to carry out the restriction digestion separately for insert a vector,so what we need to do is first we take a vector, then we add the restriction enzymebuffer which is needed by the enzyme to carry out the digestion.We also add water to make up the volume and in the last step, we add the enzyme, so enzymeusually is added in a very small quantity, so I will use a different pipette for thatso, in this case we are using EcoR1 enzyme, so throughout the process we need to makesure that it is carried out using aseptic conditions, so the tip should we autoclaved,tubes also should be sterile, at the same time we should carry out the entire reactionon ice.So, right now I have made the cocktail restriction digestion cocktail for the vector, I am goingto do the same thing for the insert and then we will keep the tubes at 37 degree whichis the optimum temperature for the enzymes to act.So, right now I am carrying a restriction digestion for the insert, so this is the insert,I am going to add the restriction buffer, so I am adding different volumes for eachof the components.And accordingly, I am adjusting the pipette as well, now I am adding water, so the lastthing is the enzyme, so I am going to vortex the tubes, so that the components are mixedproperly, then I am going to give them a short spin, so that whatever has stuck to the wallswill come down.Now, we will incubate the tubes at 37 degree for 1 hour.Now we will incubate the tubes at 37 degree.So, the incubation will be done for 1 hour, so after restriction, digestion has been performed.The next step is ligation of the 2 DNA’s, so we have our vector DNA as well as the insertDNA which has been cleaved by the restriction enzyme, so now we will join the 2 compatibleends using the ligase enzyme.So, here we have all the components of the reaction, first we will take rDNA, vectorDNA, then we will add the insert, so once we have added vector as well as insert, wewill add ligation buffer, so before adding anything, so we just need to pipette it alittle, you need to mix it a little bit.So, this is the ligation buffer, I will give it a small vortex, I will add this to theligation makes now, the last thing to be added is the ligase enzyme again, we are using asmall pipette because we require very little quantity of the enzyme, I will give it a shortvortex again to the enzyme, now our ligation mix is ready, we will vortex it a little,followed by a short spin.Now, we will incubate this at room temperature for 3 hours.After ligation is done, the next step is transformation, so after ligation the vector and the insertthey form a covalently closed structure and this DNA molecule, the combined DNA moleculewe introduce into a host cell, so in this case you have taken DH5 alpha host cell, whichdoes not have any restriction enzyme, which will chop of any foreign DNA, so we will introduceour DNA into this host cell, so that it can propagate further.So, this step transformation is carried out inside the laminar hood under a sterile conditions,so inside the hood, we have sterile filters, which may keep the inside air clean.So, before we start with the experiment, first we switch on the UV for 10 minutes, this stepensures that inside the conditions are extremely sterile, this step ensures that inside thelaminar hood conditions are extremely sterile under good for a work.So, after this we will switch off the UV and we switch on the fan and the light button.Now, we will carry out the transmission experiment, so we have our ligated sample, we will alsokeep a negative control water, so in all the experiments, we normally keep a negative control,so we have a competent cells which are the host cells in which we will introduce ourrecombinant molecule, in one of the tubes, we will add our recombinant molecule, in theother tube, we will add a negative control, water.This is our ligation mix which we will add in the competence cells, so now we will incubatethe tubes on ice for 30 minutes.Now, we will give the samples, heat shock treatment, so we will keep tubes at 42 degreeCelsius for 2 minutes.This heat shock treatment helps the host cell to get the foreign DNA this heat shock treatmenthelps the foreign DNA to get introduced inside the host cell.So, now we will perform the plating, so as we can see, we already have plates LB agarplates.These plates, they have a lot of components, which will help the bacteria to grow additionally,we have added the antibiotic ampicillin because a vector, the plasmid DNA has ampicillin resistantgene, so that when we will plate our ligated product onto this, only those colonies willshow up which have our vector.So, for 1 plate, I will be using the ligated sample which has been transformed into theDH5 alpha host cell.For the other plate, I will use the negative control which has been given the same treatmentas our ligated product, so I will take the sample, so I will add the sample in drops,so this is the spreader which I am going to use for plating, so this spreader is alreadydipped in ethanol, so that all the microorganisms if any will get killed, so I will put it toflame, so that the alcohol evaporates, I will wait for the spreader, spreader’s temperatureto get down.Because if it is too hot, it may kill my DNA sample as well, I can touch it here, so thatit becomes cool now, I will start plating, so I will be doing the plating in circularmotions, so that the sample is spreaded uniformly, I will do it till a point where I feel thatthe entire sample is spread uniformly and when the agar seems to look a little dry andthere is some friction at that point I will stop.One needs to ensure that we do not presser too hard else we may end up breaking the agarplate as well, I will show the lid some flame and I will keep it back, I will label my plate.Next, I will plate the negative control sample which is nothing but water, so normally wekeep the plates this way upside down otherwise, if we keep them like this, they sometimesbecause of the loose ends, we may end up having some sort of contamination, so usually wekeep upside down.Right now, I have done plating, so for some time I will keep it like this, straight way,later I will keep it upside down as well.Now, I will take my now I will take the negative control sample again, I will add in the formof little drops, spread it uniformly, I will show my spreader flame, I will wait for fewseconds for the spreader, for the spreader’s temperature to come down, this is how my platelooks like.Now, I have started feeling some friction on the plate, so if I poke it further, itmay break, so at this point, I will stop, now I will take my plates and I will keepthem in the incubator.So, these are the plates which I will keep in the incubator for overnight incubation,so as you can see the temperature ranges from 37 to 38 which is ideal for the bacteria togrow.I can show you another plate, which we have already done transformation yesterday andhow a transform plate looks like.So, this is a transform plate which has a lot of colonies inside, so this in this caseof transformation efficiency has been very good.In some cases, we do not get as many colonies, so right now, we have kept 2 plates in one,we hope that we have a good transformation efficiency and in the second plate, we shouldexpected to be blind because at it is just water to ensure that our experiments are absolutelyfine and there is no contamination.So, once you have obtained the colonies we need to be sure whether these colonies reflectedto recombinant molecule because as you know once we have done restriction digestion, wecan get different kinds of products.It may happen that during ligation, the vector itself ligates and have a self ligated moleculeor it may happen that there is only the insert that has transformed into the host cell, sowe need to be sure whether these colonies actually have a recombinant molecule, so onething we are sure that there is no insert here because the vector has antibiotic resistanceseen and here, in the media we have an antibiotic ampicillin.So, only those colonies which have the vector will grow on these, now those colonies canbe the self ligated vector or they can be the recombinant molecule having the insert,for that we will take these colonies, so for that we will take these colonies, we willgrow them, we will isolate the plasmid and we will check their size, so this is the LBmedia, this is the same media which has been, which is already you know in the solidifiedform in this agar plate.So, this LB media has all the components, which is required by the bacteria to grow,so we will pick up a colony, we will added in these LB media, we will also add our antibiotic,the antibiotic for which the resistant gene is already present on the vector, so thatonly our vector grows, no other contamination is seen.So, we will first add the antibiotic this is our antibiotic ampicillin, everything weare doing near the flame, so that there is no contamination, there is no chance of anycontamination now, I will pick up a colony, this again we will incubate at 37 for overnightalmost 16 hours.So, this is the shaking incubator in which I will keep this tube for overnight incubation.So, you can see the temperature set is 37 which are ideal for the bacterial growth.So, this is how a bacteria culture looks like, you can see it is very turbid and after thisis a sign that the bacteria has grown properly overnight, so this is the tube which we havejust kept for the overnight incubation, you case see the colour of the tube, it is, itis not so turbid, it is transparent but after the grow this is how it looks like.So, in conclusion, I hope now your concepts for the gene cloning is much better and muchclearer now.As I have just seen the lab demonstration session with the advent of technological advancement,the genetic engineering can be performed in a laboratory set up with great ease now, whyit might you know look a straightforward but the cloning a gene often involves you knowlot of thought process and the whole parameter optimisation may take several months timeto really obtain the correct clone.And then you have to ensure that you know the sequence is correct and it is not self-ligatedvector, what you obtain is the right gene of interest which you started with, so ofcourse, the process is straightforward and much simpler but to actually obtain a rightclone in frame does need you know good listener operating protocols and sometime you knowgood experience of knowing the concepts involved in genetic engineering.So, I hope you enjoyed the lab session and we will continue our interactions and discussionabout gene cloning in the next lecture as well, thank you.

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Picking up Plasmid DNA using Nanodrop, but not using Electrophoresis - Biology






The First International Workshop at

Cairo University Research Park
Application of Genetic Engineering in Biotechnology
Genetic engineering techniques have been applied in numerous fields including research, agriculture, industrial biotechnology, and medicine. Enzymes used in laundry detergent and medicines such as insulin and human growth hormone are now manufactured in GM cells, experimental GM cell lines and GM animals such as mice are being used for research purposes, and genetically modified crops have been commercialized.
It is a pleasure to announce that Cairo University Research Park (CURP) will launch a Genetic Engineering Workshop to attract applicants with highest intellectual and scientific skills in the area of biology. This workshop combines the opportunities to perform the latest, as well as commonly used molecular biology techniques for cloning of genes, sequencing, and sequence analysis with gene expression particular attention to the principles underlying each technique.
This workshop will be held from Aug 17th to Sep 5th, 2013 and carried out by the Molecular Genetics Group at Cairo University Research Park.
This workshop's hands-on activities were including:
· Isolation of total RNA and purify the mRNA.
· Construction of cDNA library.
· Gene cloning: specific gene and cDNA library construction.
· Cloning of PCR product in bacterial plasmid.
· Transformation in bacterial cells.
· Colony pick up and colony PCR.
· DNA plasmid extraction.
· Digestion DNA with restriction enzymes.
· DNA gel electrophoresis and data analysis.
· Isolation of novel tissue-specific genes from cDNA libraries.
· DNA sequencing & DNA fingerprinting.
· Gene expression analysis using microarray.
· Single-cell analysis of gene expression by fluorescence microscopy.
· Genomics and bioinformatics.
· Protein expression analysis (Western Blotting and ELISA).
No previous experience in molecular biology is required or expected. Twenty participants will be selected from a variety of disciplines and academic backgrounds, including principal investigators, directors of programs, medical doctors, postdoctoral fellows, graduate students, research assistants, sales associates, equipment engineers, etc.
The three-week long courses cover in-depth DNA cloning, PCR, DNA sequencing, genomics and bioinformatics and gene expression. Learn hands-on techniques used in gene expression analysis including, quantitative RT-PCR, bioinformatics and genomics and protein expression.
Registration deadline is Thursday, July 20th 2013.
Enrollment is limited to 20 participants.
Registration is open to all Nile River and Middle East Countries. Notification of acceptance will be emailed on July 22nd, 2013. Please register only if you are able to attend the entire workshop. Registrants must mail in a reservation check for $100 to guarantee space in the workshop. It will be held and returned at the workshop, but will be forfeited for no-shows. Mail checks to: Faculty of Agriculture Bank Account. You will not be considered for attendance if we do not receive your check.



Organizer:


Picking up Plasmid DNA using Nanodrop, but not using Electrophoresis - Biology

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For the Student:

1. Pick up two clean microfuge tubes. Label one “P+” and the other “P-.”

2. Pick up a Styrofoam cup with crushed ice and place one tube containing 100 μL of competent cells into the ice. It’s important that the cells remain at 0°C. Also, place your P+ and P- tubes into the ice.

3. Pick up your ligated plasmids from the microfuge tube rack labeled “LIG tubes.” Your “LIG” tube should be labeled with your group number and class period.

4. Set the P-200 pipettor to 50 μL (set to “0-5-0”) and place a clean tip onto its barrel. Very carefully resuspend the cells by gently pumping the cells in and out two times. Hold the tube by the upper rim to avoid warming the cells with your fingers.

5. Aliquot 50 μL of the resuspended cells into the prechilled P+ and P- microfuge tubes. Immediately return the aliquoted cells to the wet ice. Hold the tubes by the upper rim to avoid warming the cells with your fingers.

6. Using the P-20 pipette, add 10 μL of your ligated plasmid to the tube labeled “P+.” Gently mix the plasmid with the cell suspension by pumping the cell suspension two times. Immediately return the P+ tube into the ice. Do not add plasmid to the P– tube. The cells in this tube will serve as the “plasmid control.”

7. Keep the cells in ice for 15 minutes.

8. While the cells are incubating in ice, obtain the following: One each of these agar plates: LB, LB/amp (LB + ampicillin) and LB/amp/ara (LB + amp + arabinose)

9. Label the bottoms (plate containing the agar) of all three plates with your group number and class period. Write small and on the edge of the plate. Then divide the LB and amp plates down the middle using two lines. Label one half of each plate “P+” and the other half with a “P-.” See the diagram below. Do not divide the ara plate.

10. Following the 15-minute incubation in ice, carry the ice cup containing the cells to the 42°C water bath. Take the tubes from the ice and hold them in the water bath for 45 seconds. After the 45-second heat shock, place them back into the wet ice immediately for at least one minute.

11. After one minute, use the P-200 pipette to add 150 μL (set to “1-5-0”) of LB broth to the P- tube. Cap the tube and gently flick the lower portion of the tube two or three times to mix.

12. Use a new tip and transfer 150 μL of LB broth to the P+ tube. Close the cap and gently flick the tube to mix.

13. Obtain one package of sterile cell spreaders from your teacher. You are now ready to spread your bacterial cells onto the sterile agar plates.

14. Using the P-200 pipette (set to “0-5-0”), gently pump the pipette two or three times to resuspend the cells then aspirate 50 μL of cells from the P- tube. Open the lid from the LB plate like a “clamshell.” Dispense these cells on the half of the plate marked “P-.” Close the lid.

15. Resuspend the cells by gently pumping the pipette then aspirate a second 50 μL aliquot for the LB/amp plate. Remember, you want to deposit the P– cells on the half of the plate you labeled “P-.” Cover the plate.

16. Open the package of sterile cell spreaders at the end closest to the spreader handles. You will share this package with another group. Remove only one spreader, keeping the others sterile. Hold the spreader by the handle and do not allow the bent end to touch any surface, as this will contaminate the spreader. Close the package to avoid contaminating any of the other spreaders.

17. Open the lid to the LB plate, like a clamshell, and gently using a light, gliding motion spread the cells across the surface of the agar, keeping the cells on the P– side of the plate. Try to spread them evenly and along the sides of the plate as well. Carefully spread the P- cells on the LB/amp plate using the same spreader and technique. Place the used spreader into the biohazard bag.

18. Repeat steps 14 thru 17 to inoculate the LB and LB/amp plates with the P+ culture. Be certain to use the “+” pipette and a new spreader to avoid contamination.

19. Now you’re ready to inoculate the LB/amp/ara plate! Using the P-200 pipette (set to “1-0-0”), transfer 100 μL of the P+ culture onto the surface of the LB/amp/ara plate. Deposit the 100μL of cells on several areas across the agar surface rather than a single spot. Lift the lid, clamshell style, and spread the cells evenly over the surface of the plate. Gently rotate the plate beneath the P+ spreader so that the cells can be spread over the entire surface of this plate. Try to get the cells spread along the wall of the plate as well. Cover the plate when finished.

20. Allow all three plates to sit right-side up for five minutes.

21. Using colored tape, tape all three plates together and place them in the incubator, gel-side up. Be certain that you have clearly labeled your plates with your group number and class period. You can mark the tape to help you find them for the next lab.

22. Discard cell-contaminated waste: spreaders, cell tubes, pipette tips, by placing them into the cell-contaminated waste bag provided by your teacher.


Kandace's Biology Blog

Questions Regarding GATTACA!
1. The following terms were used in the movie. How do they relate to the words we use: degenerate and invalid?
De-gene-erate: What a god child is called
In-valid: The type of person that wasn’t made
Borrowed Ladder: A person that you disguise yourself as

2. Why do you think Vincent left his family, tearing his picture out of the family photo, after winning the swimming race against his brother?
I think he finally left the family so he could start his life over in a sense and prove his point to his brother that he could do whatever he set his mind too even though he was different. However after he finished that he seemed to have the strength to move on with his life, and finish things he thrived to do.

3. Describe the relationship between Vincent and Anton.
Vincent and Anton, were brothers in this movie. They were extremely competitive with one another. Both of the two were always trying to be better than the other at multiple things. Anton just happened to be the detective in Vincent's case which added to this sibling rivalry.

4. When Jerome Morrow said to Vincent/Jerome, “They’re not looking for you. When they look at you, they only see me,” what did he mean? Can you find any parallels to this type of situation in real life?
What he meant by this situation was that technically speaking Vincent no longer existed now that Jerome had become what Vincent once was. Therefor no one realized that Jerome was around because the connection between the two was never made. Also this conversation could have meant that being accused of things would no longer happen to Vincent because his record show him as another person. I would say that the parallel in this situation is when Jerome hears something about his old self. He would more than likely become nervous because he is still the same person overall.

5. Choose your favorite character from the film. Explain why you choose that person. Would you want to be that person? Why? Why not?
My favorite character in GATTACA would have to have been Vincent. In my opinion he was the most prominant character with an idea and a mission, which added to the excitement of his character. Also he was very determined to accomplish certain activities and that drive was a great characteristic to me. However I did feel bad for him since he had to change his identity.

6. At the end of the film, you are told that the Doctor knew about Vincent all along. Why did the Doctor go along with the fraud? What would you have done if you were the Doctor?
I would have to say that the doctor went along with the deception the whole time because he was on Vincent's side and he wanted him to accomplish what he set out to do. Personally I would probably have went along with it as well because I wouldn't want to rat someone out that had worked so hard to be an achiever.

7. The technology to do what was done in the movie is definitely possible within the next fifty years. Do you think that Vincent’s world could eventually happen in America? Why?
I don't think this would necessarily happen in America because we have rights within the government. I'm sure throughout the years this process could happen, especially with how fast our technology changes, so I wouldn't doubt it occurring, but I don't believe it will be that popular in America.

8. What do you think is wrong with the society portrayed in "GATTACA"? What is right?
I don't believe anything was necessarily right in this movie. The whole point of the parents choosing how the baby would be was in my opinion wrong. You should love the baby you conceive without changing it. The discrimination that was presented against the "invalid" ones, was ridiculous to me. After all nothing is perfect. To me it seemed like if you weren't perfect than you weren't good enough and I really disliked that situation.

9. What were the screenwriters trying to tell us through the episode of the 12-fingered pianist? Is anything wrong with engineering children to have 12 fingers if, as a result, they will be able to make extraordinarily beautiful music?
I believe the screenwriters were trying to say that people would start trying to engineer children to be good at certain things and succeed in them, which is obviously wrong because its what the parents want not the children.

10. You and your spouse are having a child and are at the Genetic Clinic pictured in the movie. What characteristics would you want for your child and what would you ask to be excluded? Why would you make those choices?
Normally I woul say that there is no way I would go to a clinic sugh as this because of the ways they discriminate against babies that are yet to even be born. But if I had to choose I would want them to be very athletic, tall and smart. However I would take out my families "mole" gene from the sequence.

11. Picture yourself as either Vincent, Jerome, or Anton. Would you have acted the same or done things differently if you were in the same world as them?
I probably would've acted the same as they all had. Each of them handled each situation in the best way that they could and I don't think anyone should doubt them about their actions. However I would not have committed suicide like Jerome did.


12. How does the society in GATTACA resemble the type of society America was during the height of the eugenics movement?
People were extremely anxious and excited about being able to create their own baby and help make them "better" in their eyes which is just like in GATTACA. However in GATTACA the clinic babies were the only ones who were accepted for the eugenics experiment.

Overall I enjoyed the parts and pieces I seen to the GATTACA movie. It was a very different movie, but it taught me a lot about eugenics. However I hope America never turns into a country where we create our children instead of just conceiving them naturally.


Watch the video: How to Quantify DNA with a Spectrophotometer (May 2022).


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