I am trying to use the overlap extension PCR to combine two linear fragments of approximately 1200 base pairs in size. My first SOE-PCR was successful using Taq polymerase, with annealing and overlap temperatures set at 60 degrees Celsius. It had smear with my desired sharp bond. after that when I trying to repeat the process, I only obtained a smear with no specific bonds.
I amplified my fragments with taq and also pfu, but I don’t get my desired bond. I had just smear.
Does anyone have such experiment and help me, please?
There are a few things you may try:
- if you have unspecific amplification in the first PCRs, try to purify the linear segments you want to use.
- check the annealing temperature of the overlapping sequence (calculate the melting temperature - there are a few online tools for this or simply use the formula: Tm = (amount of G/C * 4) + (amount of A/T * 2)); and then use 5^C below that as your annealing temperature).
- if not used, add end primers (of the desired final fragment) to the PCR.
- if 1.2 Kbp is the length of your final fragment, use 1,5 min as your extension time.
I hope these are helpful.
Hello,
Recently I used this Overlapping PCR strategy. it worked for me.
-I am in support with all the comments provided by-Jonny Yokosawa.
Additionaly i want to add on
1) while doing the OE-PCR with both fragments the stochiometry of each fragment is important. and keep less cycles (13-15) in step 2.In this stage omit the primers in the reaction.
2) Do a PCR clean up for the OE-PCR product and keep a nested PCR to amplify entire fragment by adding the end primers.
i hope these will be helpful. all the best.
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