I’m able to see colonies on a plate, but when I pick them for liquid culture, no growth is observed. Why?

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Answer

One possible explanation could be toxicity associated with the insert. This toxicity does not affect slow growing cells on solid medium but is much stronger in faster growth conditions like liquid medium.

Suggestions:
1. Use TOP10F’ or any other strain with the LacIq repressor
2. Try using any other strain appropriate for cloning.
3. Lower growth temperature to 27 - 30 degrees C and grow the culture longer
4. Another possibility to explain lack of growth is possible phage contamination. In this situation we recommend using an E. coli strain that is T1 phage resistant like DH5alpha-T1R.

Answer Id: E6669

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I see small satellite colonies on my LB+Amp plates. Why is this?

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Answer

These small colonies are most likely caused by degradation of the Ampicillin. The colonies are just untransformed cells that grow on LB with degraded Amp. In order to circumvent this scenario, you can try to:
1. Plate cells at a lower density
2. Use fresh LB-Amp plates or replace Ampicillin with carbenicillin.
3. The plates should not be incubated for more than 20 hours at 37 degrees C. Beta-lactamase, the enzyme produced from the Ampicillin-resistance gene, is secreted from the Amp-resistant transformants and inactivates the antibiotic in the area surrounding the transformant colony. This inactivation of the selection agent allows satellite colonies (which are not truly Amp-resistant) to grow. This is also true if carbenicillin is being used.

Answer Id: E6670

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What considerations should I take into account when designing primers for PCR of an insert which will be cloned into a TOPO™ vector?

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Answer

PCR primers should not have 5’-phosphates when cloning into any TOPO™ vector, as the presence of 5’-phosphates inhibit the TOPO™ cloning reaction. Phosphorylated products can be TA-cloned but not TOPO-cloned. This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. TOPO™ vectors have a 3’ phosphate to which topoisomerase is covalently bound and a 5’ phosphate. Non-TOPO™ linear vectors (TA and Blunt) have a 3’ OH and a 5’ phosphate. Phosphorylated products should be treated with phosphatase (CIAP) prior to TOPO-cloning. Treatment with CIAP may raise efficiency to 25%. PCR products generated with 5’-biotinylated primers (or any other 5’-label including 5’-Cy5) will not ligate into any of the TOPO™ vectors due to steric hindrance.

Answer Id: E6746

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What are some of the prerequisites for TOPO™ cloning?

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Answer

Please consider the following before TOPO™ cloning:

- TOPO™ cloning cannot ligate DNA with a 5’ phosphate group.
- TOPO™ cloning will decrease in efficiency inversely with the size of the insert (above 3 kb) unless using the TOPO™ XL cloning kit.
- TOPO™ vectors contain different antibiotic resistance markers which should be considered before purchase.
- TOPO™ TA vectors accept fragments containing a 3’ A overhang while Zero Blunt™ vectors accept fragments that are blunt-ended.

Answer Id: E6736

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What can inhibit the TOPO™ cloning reaction?

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Answer

When doing a TOPO™ cloning reaction, 2 μl of a PCR reaction containing up to 10% DMSO or 1.3 M betaine will not interfere with the TOPO™ reaction. Formamide and high levels of glycerol will inhibit the reaction. These reagents are usually added to the PCR reaction to enhance the yield of the PCR product, e.g., to reduce the effect of secondary structure or assist in amplification of GC-rich sequences. The effects of tricine or acetamide have not been tested on the TOPO™ cloning reaction.

Answer Id: E6747

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What range of PCR product (molar ratios and ng quantities) do you suggest for TOPO™ TA cloning?

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Answer

We suggest starting with a molar ratio of 1:1 (insert:vector), with a range of 0.5:1 to 2:1 (insert:vector). The ng quantities should be between 5-10 ng of a 2 kb PCR product in a TOPO™ cloning reaction.

Answer Id: E6737

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What suggestions can you make for blue/white screening?

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Answer

1. Use pUC or pUC-based vectors that contain the portion of the lacZ gene that allows for ? complementation.
2. Select an E. coli strain that carries the lacZdeltaM15 marker.
3. Plate transformations on plates containing X-gal. Spread 50 μg of 2% X-gal or 100 microliters of 2% bluo-gal (both can be dissolved in DMF or 50:50 mixture of DMSO:water) on the surface of a 100 mm plate and let dry. Alternatively, add directly to the cooled medium (~50 degrees C) before pouring the plates at a final concentration of 50 μg/mL for X-gal and 300 μg/mL for bluo-gal. Plates are stable for 4 weeks at 4 degrees C.
4. If the strain used carries the lacIq marker, add IPTG to induce the lac promoter. Spread 30 μl of 100 mM IPTG (in water) on 100 mm plates. Alternatively, add the IPTG directly to cooled medium (~50 degrees C) before pouring the plates to a final concentration of 1 mM. Plates are stable for 4 weeks at 4 degrees C.
5. Do not plate E. coli on medium containing glucose if using X-gal or bluo-gal for blue-white screening. Glucose competes as a substrate and prevents cells from turning blue.

Answer Id: E6716

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I’m transforming pCR™2.1-TOPO™ clones into TOP10F’ cells. Will I need to add IPTG to my plates along with X-gal for blue/white screening? What if I used TOP10 cells instead?

Product FAQ

Answer

The F’ episome in TOP10F’ has a lacIq marker, which over-expresses the lac repressor. IPTG must be added to LB plates along with X-gal to see beta-galactosidase expression and blue color in this strain. TOP10, on the other hand, does not require IPTG for blue/white screening.

Answer Id: E6727

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I’m getting low cloning efficiency with my TOPO™ cloning reactions. What should I do?

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Answer

Please consider the following possible causes:
- pH > 9: Check the pH of the PCR amplification reaction and adjust with 1 M Tris-HCl, pH 8.
- Excess (or overly dilute) PCR product: Reduce (or concentrate) the amount of PCR product.
- Incomplete extension during PCR: Be sure to include a final extension step of 7 to 30 minutes during PCR. Longer PCR products will need a longer extension time.
- Cloning large inserts (>1 kb): Try one or all of the following suggestions: Increase amount of insert. Incubate the TOPO™ ™ cloning reaction longer. Gel-purify the insert using either a silica-based DNA purification system (e.g., PureLink™ system) or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)
- PCR product does not contain sufficient 3’ A-overhangs even though you used Taq polymerase: Increase the final extension time to ensure all 3’ ends are adenylated. Taq polymerase is less efficient at adding a nontemplate 3’ A next to another A. Taq is most efficient at adding a nontemplate 3’ A next to a C. You may have to redesign your primers so that they contain a 5’ G instead of a 5´ T.

Answer Id: E6763

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What is the difference between a stop solution and salt solution? What is its function in the TOPO™ kit?

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Answer

The composition of the 6X Stop solution is 0.3 M NaCl, 0.06 M MgCl2, and the composition of the 6X Salt solution is 1.2 M NaCl, 0.06 M MgCl2. Stop solution is only included in the TOPO™ XL Cloning kit whereas Salt solution is currently included in all of the other TOPO™ cloning kits. These solutions prevent free topoisomerase from re-binding and nicking the plasmid, which would reduce the number of colonies from a TOPO™ reaction.

Answer Id: E6748

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I’m trying to transform large plasmid, 40 kb in size. What strain should I use?

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Answer

While there is no specific strain that works better with large plasmids, it is important to focus on transformation efficiency. For larger plasmids, chemically competent cells with highest efficiency are suggested, such as OmniMAX™ 2, TOP10, etc. We would recommend using an electrocompetent cell strain with plasmids larger than 20 kb for best efficiency.

Answer Id: E6730

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I typically store my PCR products before TOPO™ cloning. Is this okay?

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Answer

For optimal TOPO™ cloning, we recommend using fresh PCR products.

Answer Id: E6744

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I’m getting no colonies at all on my plates. Can you help?

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Answer

We recommend trying the following:
- Carry out the puc19 transformation control; this gives you information about the performance of the cells.
- Check plates for expiration and correct media used (LB/agar).
- Confirm that the correct antibiotic and concentration was used.

Answer Id: E6728

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I’m getting no colonies after transformation. What should I do?

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Answer

No colonies may occur due to the following problems:

Bacteria were not competent. Use the pUC18 vector included with the One Shot™ module to check the transformation efficiency of the cells.
- Incorrect concentration of antibiotic on plates, or the plates are too old. Use 100 μg/mL of ampicillin or 50 μg/mL kanamycin. Be sure ampicillin plates are fresh (< 1 month old).
- The product was phosphorylated (TOPO™ cloning only). Phosphorylated products can be TA-cloned but not TOPO-cloned. This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. The TOPO™ vector has a 3’ phosphate to which topoisomerase is covalently bound and a 5’ phosphate. The non- TOPO™ vectors (TA and Blunt) have a 3’ OH and a 5’ phosphate. Phosphorylated products should be phosphatased (CIP) before TOPO-cloning.

Answer Id: E6764

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I’m able to get a lot of colonies, however, none contain my insert of interest. What should I do?

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Answer

You may be cloning in an artifact. TA and TOPO™ Cloning are very efficient for small fragments (< 100 bp) present in certain PCR reactions. Gel-purify your PCR product using either a silica-based DNA purification system or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)

Answer Id: E6766

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