Q: Can PCR products generated with GoTaq® DNA Polymerase be used to for T- vector cloning?
A: Yes. GoTaq® DNA Polymerase is a robust formulation of unmodified Taq Polymerase. GoTaq®DNA Polymerase lacks 3’ →5’ exonuclease activity (proof reading) and also displays non-template–dependent terminal transferase activity that adds a 3′ deoxyadenosine (dA) to product ends. As a result, PCR products amplified using GoTaq® DNA Polymerase will contain A-overhangs which makes it suitable for T-vector cloning.
Q: Can GoTaq® Long PCR Master Mix be used for T-Vector Cloning?
A: Yes it can. GoTaq® Long PCR Master Mix utilizes recombinant Taq DNA polymerase as well as a small amount of a recombinant proofreading DNA polymerase. This 3´→5´ exonuclease activity (proof reading) enables amplification of long targets. Despite the presence of a small amount of 3´→5´ exonuclease activity, the GoTaq® Long PCR Master Mix generates PCR products that can be successfully ligated into the pGEM®-T Easy Vector System.
We have demonstrated that GoTaq® Long PCR Master Mix successfully generated DNA fragments that could be ligated into pGEM®-T Easy Vector System without an A-tailing procedure, and with ligation efficiencies similar to those observed with the GoTaq® Green Master Mix.
For details refer to Truman, A., Hook, B. and Wieczorek, D. Using GoTaq® Long PCR Master Mix for T-Vector Cloning.
Tip: For cloning blunt-ended PCR fragments into T-vectors, use the A-tailing protocol discussed in the pGEM®-T and pGEM®-T Easy Technical Manual #TM042.
Q: How do I prepare PCR products for ligation? What products can be used to purify the DNA?
A: The Wizard® SV Gel and PCR Clean-Up System (Cat.# A9281) can be used to purify GoTaq® Polymerase-generated PCR products prior to T-vector cloning. It may be possible to successfully clone PCR products directly without prior purification in cases where a single amplification product is generated. However, even if no extraneous bands are visible, there may be some primer-dimers present in the reaction, and this can result in unacceptably high numbers of clones containing primer-dimer instead of the fragment of interest.
Q: What factors affect transformation efficiency? How do I determine the efficiency of my competent cells?
A: Ligation of fragments with a single base overhang can be inefficient, so it is essential to use cells with a transformation efficiency of at least 1 × 108cfu/µg DNA in order to obtain a reasonable number of colonies.
Here is how one can determine the efficiency of the competent cells used for transformation.
A 100μl of competent cells are transformed with 0.1ng of uncut plasmid DNA. Any standard plasmid of known concentration can be used for this purpose. The transformation reaction is mixed with 900μl of SOC medium. This results in a final concentration of 0.1ng DNA/ml. This transformation mix is diluted 1:10 with SOC medium which results in 0.01ng DNA/ml and 100μl (0.001ng DNA) of the dilution is spread on replicate plates.
Assuming that an average of 200 colonies are obtained the transformation efficiency is calculated as
200cfu ÷ 0.001ng = 2 × 105cfu/ng = 2 × 108cfu/μg DNA
If you are using competent cells other than JM109 High Efficiency Competent Cells purchased from Promega, it is important that the appropriate transformation protocol be followed. Selection for transformed host cells should be on LB plates with the appropriate antibiotic and IPTG/ X-Gal added at the appropriate concentrations.
Tip: For best results, we recommend to use plates that are less than 1 month old.
Q: How do I screen for recombinants in T-cloning? Why do my recombinants appear blue?
A: Potential recombinants can be chosen by an initial blue/white colony screen. Recombinants may be analyzed further by techniques such as restriction enzyme analysis or small-scale PCR screening.
Blue/white colony screening relies on disruption of the lacZ gene. Although the pGEM®-T Vector Control DNA will produce recombinants that generate white colonies, insertion of other DNA fragments into the lacZ coding sequence may not result in white colonies unless the fragments disrupt the lacZ reading frame. Although this tends to occur most frequently with PCR products of 500bp or less, inserts of up to 2kb have been reported to result in blue colonies. Moreover, some insert DNAs can also result in pale blue colonies. For this reason, we recommend performing a control ligation without insert DNA. For instance, the control ligation without Control DNA may produce 20–40 blue colonies, while the experimental ligation produces 100 blue colonies. This result strongly suggests that the insert of interest has been cloned but has not sufficiently disrupted the lacZ coding sequence.
Tip: Blue color will become darker after the plate has been stored overnight at 4°C.
For more information on these methods and the topic of cloning, read the Cloning chapter of our Protocols and Applications Guide. As always, Promega Technical Services Scientists are here to assist you in troubleshooting your experiments at any time. Contact Technical Services.