PCR Cloning Support - Troubleshooting

No, unfortunately not. Electrocompetent cells are not chemically treated. Unlike chemically competent cells, electrocompetent cells have an intact cell membrane.

To increase the intensity of the crystal violet-stained gel, allow the gel to lie for a couple of hours in crystal violet stain (45 µL of 2 mg/mL stain in 100 mL sterile water or 1-10 µg/mL stain in 0.1X TAE) and it will stain a little darker. Place the gel over a white background when excising the band to improve visibility.

The ends of pCR™II and pCR™2.1 vectors are palindromic sequences, since both ends must accommodate the TOPO^® binding sites. Therefore, there is an inherent potential for secondary structure once the vector is ligated. This does not usually affect sequencing or PCR results, as cycling conditions are usually sufficient to overcome potential secondary structures that might form. However, certain inserts might enhance the formation of such secondary structures by the regions flanking the cloning site. Strong secondary structures may inhibit the cycle sequencing reactions commonly used in automated sequencing. If you suspect this is a problem for your clone, here are a few things to try:

1. Increase the annealing temperature of the cycling, or even try a two-stage cycling reaction.
2. Add DMSO to the reaction. This may reduce secondary structure formation. However, beware that it may also affect the fluorescence or some other portion of the reaction.
3. Design primers internal to the insert.
4. Last resort: switch to non-cycling conditions, e.g., manual sequencing using sequenase, S-35-labelled dNTPs. This is perhaps the least convenient, but most effective method.

Please try the suggestions below to increase the number of colonies.

• Longer incubation of the TOPO® cloning reaction at room temperature, provided that the 6X Salt solution is added to the reaction.
• Electroporation can give significant increases in colony numbers; often 10-20 fold higher. However, if doing electroporation, it is important that the TOPO® reaction mix contains diluted Salt solution or, for best results, precipitated prior to transformation. For high primary transformants by electroporation it is recommended to:
  • Add 100 µL double diH2O to the 6 µL TOPO® reaction and incubate 10 more minutes at 37 degrees C.
  • Precipitate by adding 10 µL 3 M Na-Acetate, 2 µL 20 µg/µL glycogen, 300 µL 100% ethanol. Place on dry ice or –80 degrees C for 20 min, spin at top speed in a microcentrifuge at 4 degrees C for 15 min. Wash pellet with 800 µL 80% ethanol, spin at top speed for 10 min, pour off ethanol, spin 1 min, and remove remaining ethanol without disturbing pellet. Dry pellet (air-dry or speed-vac).
  • Resuspend pellet in 10 µL ddH2O and electroporate 3.3 µL of resuspended DNA according to a normal electroporation protocol. This electroporation protocol can yield up to 20 fold more colonies than chemical transformation of an equivalent TOPO-reaction. The addition of the 100 µL ddH2O followed by 10 mins incubation is not absolutely necessary, but it sufficiently dilutes the reaction and may help inactivate topoisomerase so that it is more easily electroporated.

Here are suggestions to try to increase your cloning efficiency with dTOPO® cloning:

• Ensure that the 5’ primer has –CACC and the 3’ primer does not have sequence similarity to GTGG.
• The molar ratio of PCR product: TOPO® vector used is critical to success. We recommend using a 1:1 to 2:1 molar ratio, starting with a 1:1 of PCR product: TOPO® vector. The TOPO® cloning efficiency decreases significantly if the ratio of PCR product: TOPO® vector is <0.1:1 or >5:1. These results are generally obtained if too little PCR product is used (i.e., PCR product is too dilute) or if too much PCR product is used in the TOPO® cloning reaction. If the yield of the PCR product has been quantitated, the concentration of the PCR product may need to be adjusted before proceeding to TOPO® cloning. For pENTR™ TOPO® vectors, using 1-5 ng of a 1 kb PCR product or 5-10 ng of a 2 kb PCR product in a TOPO® cloning reaction generally results in a suitable number of colonies.

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.

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.

There could be a few possibilities for this:

• The insert does not interrupt the reading frame of the lacZ gene. If you have a small insert (< 500 bp), you may have light blue colonies. Analyze some of these blue colonies as they may contain insert.
• A polymerase that does not add 3´ A-overhangs was used. If you used a proofreading enzyme, you will need to do a post-reaction treatment with Taq polymerase to add the 3’ A-overhangs.
• PCR products were gel-purified before ligation. Gel purification can remove the single 3´ A- overhangs. Otherwise, optimization of your PCR can be performed so that you can go directly from PCR to cloning.
• The PCR products were stored for a long period of time before ligation reaction. Use fresh PCR products. Efficiencies are reduced after as little as 1 day of storage.
• Too much of the amplification reaction was added to the ligation. The high salt content of PCR can inhibit ligation. Use no more than 2-3 μl of the PCR mixture in the ligation reaction.
• The molar ratio of vector:insert in the ligation reaction may be incorrect. Estimate the concentration of the PCR product. Set up the ligation reaction with a 1:1 or 1:3 vector:insert molar ratio.

On a typical plate there are a few white colonies which do not contain insert. These are usually larger than the other colonies and are due to a deletion of a portion of the plasmid sequence by a rare recombination event (usually from the polylinker to a site in the F1 origin). To find a colony with an insert it is best to pick clones of a variety of color and pattern for analysis. Often an insert will generate two distinct patterns according to its orientation.

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.)

If the insert is potentially toxic to the host cells, here are some suggestions that you can try:

• After transforming TOP10 or DH5α cells, incubate at 25-30 degrees C instead of 37 degrees C. This will slow down the growth and will increase the chances of cloning a potentially toxic insert.
• Try using TOP10F’ cells for the transformation, but do not add IPTG to the plates. These cells carry the lacIq repressor that represses expression from the lac promoter and so allows cloning of toxic genes. Keep in mind that in the absence of IPTG, blue-white screening cannot be performed.
• Try using Stbl2 cells for the transformation.

Here are possible causes and suggestions:

• Incorrect PCR primer design: Make sure that the forward PCR primer contains the sequence, CACC, at the 5′ end. The 4 nucleotides, CACC, base pair with the overhang sequence, GTGG, in the Directional TOPO® vector.
• Reverse PCR primer is complementary to the GTGG overhang at the 5′ end: Make sure that the reverse PCR primer does not contain the sequence, CACC, at the 5′ end.
• Insert cloned in the correct orientation is potentially toxic resulting in cloning in the reverse orientation: After transformation, incubate the cells at 25-30 degrees C instead of 37 degrees C. This will slow down the growth and will increase the chances of cloning a potentially toxic insert.
• Try using TOP10F’ cells for the transformation, but do not add IPTG to the plates. These cells carry the lacIq repressor that represses expression from the lac promoter and so allows cloning of toxic genes. Keep in mind that in the absence of IPTG, blue-white screening cannot be performed.
• Try using Stbl2 cells for the transformation.

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 phosphatased (CIP) before TOPO® cloning.

Using the vector only for transformation is not a recommended negative control. The process of topo-adaptation is not a 100% process, therefore, there will be “vector only” present in your mix, and colonies will be obtained.