I have run PCR for functional genes using AccuStart Tough mix II and have tried cloning before this and I did not get any successful cloning. The manufacturer master mix manufacturer claimed that the polymerase act just like regular taq polymerase and will produce A-tailing. I'm using the master mix since our samples are quite tough to amplify and I've tried tons of polymerases and master mixes and this one works best in my previous experiments. I have narrow down the problem to one possibility: no A-tailing in my PCR products. If the master mix does produce A-tailings, I'm just wondering if storage in the fridge overnight would degrade the A-tailings from my PCR products?
A few comments here:
1. About the storage condition: Poly A tails are generally stable if handled properly. I would however store my amplicons at -20 (if I do not have time to perform the cloning on the same day).
2. About the polymerase: As per the manufacturer's (Accustart mix) instructions, the polymerase does not have a 3'-5'exonuclease activity and hence the amplicons should have a dA overhang. During your pcr, do you include a final extension step? If no, I would recommend a final extension for 7-10 minutes - this step does not really do much but complete the amplifications and add dAs.
3. About other strategies: I cannot emphasize this enough, but if the genes are so important, I would perform the cloning on the same day of the pcr. Once a pcr is performed - ligation and transformation hardly takes any time. Regardless, if you have to absolutely store the pcr products overnite and perform a cloning on the next day - and are very doubtful about the presence of a dA overhang on the next day - then you can incubate your "blunt-ended" amplicons with a routine, non-proof reading polymerase, and dATP. This should definitely do the work.
All the best.
Previously, I did the cloning the next day after the amplification. I did gel purification then the cloning step. I have tried cloning right after PCR with different samples as well and I get the same result. Very few
1. What do you mean by very few? few colonies - did u screen them?
2. After the pcr, when you ran the amplicon on the gel - did you see a clean, single band? If yes, you might not need to perform a gel purification - can proceed with directly or with a pcr purification, if necessary. Excessive purification sometimes cleaves off the overhangs.
I dunno what happened. It seems the text I wrote was cut off. I was meant to say I had very few successful positive colonies. And all the positive colonies I sent for sequencing turned to be false positive. No insert whatsoever.
I have single band on gel and I already did what you said. I have also performed cloning using both gel extracted amplicons and purified amplicons. The purified amplicons gave me higher number of successful clones but all are false positives after I sent them for sequencing. That's why I thought maybe its the polymerase. If it is indeed the polymerase that cause me the trouble, would it solve my problem if I run PCR with the same master mix with addition of taq polymerase (1U) as additive to the PCR reaction?
A few comments here:
A. What is the length of your insert?
B. What type of screening method are you using to identify your "successful clones" on your petri dish? Are you performing a blue-white screening and/or a colony pcr? If no, include them.
C. Do you have a positive & a negative control for your TA vector? Does it work - how many positive colonies do you get here?
D. What is your ligation ratio? Try altering the insert concentrations in your ligation mix.
E. Do not use taq polymerase as an additive. It may not be a very good idea of combining two different forms of polymerase in the same reaction. You can either perform reaction A and B separately or add taq, dATP, taq buffer to the same reaction mix - once the first pcr is done.
All the best.
DNA is exceptionally stable. I have stored pA overhang DNA at room temperature for multiple months and still had successful transformations. Obviously, long term storage should be done at cold temperatures. Sounds like you have one of two problems.. You are either not putting enough DNA into the ligation reaction. The more likely problem is that your competent cells or transformation protocol are not working.
Deepak
A: 552 bp
B: Both blue white screening and colony pcr. white colonies are very low and colony pcr was not successful. Then I sent for sequencing just to make sure I don't have the insert.
C: Positive and negative controls are a must. I always run them and they work well. Positive colonies range from 100-200 colonies depending on the volume I use for plating.
D: I have tried ligation ratio from 3:1 to 8:1 (insert:vector)
E: I read several protocol that combine a master mix with different taq polymerase. That's why I was thinking of trying that with my samples. I probably will try do the reaction for A-tailing addition.
Matthew,
The ratio i used for ligation range from 3:1 to 8:1 (insert:vector). And controls I prepared turned out as expected. I've used different competent cells as well and it was the same. Should I use different vector? Currently I'm using pGEMt
a. Assuming that you have followed all the steps that you have mentioned above, I would then try using the insert directly from the pcr - without purification (under the assumption that your pcr is clean).
b. I would use a ligation ratio of 1:5. Use about 25ng of vector. Also, I would setup at least 2-test ligation reactions: A. @25 degrees 1-2h B. @16 degrees overnight.
Rest of your controls would be as is.
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