Dear Colleagues,
I'm trying to synthesize a linear deletion cassettes using over-lap PCR, as described in the paper:
B. Lesic, L.G. Rahme (2008). BMC Molecular Biology 9:20
There are three steps of synthesis:
1. In the first step, gene-of-interest flanking regions and antibiotic resistance cassette are synthesized independently.
2. Next, three products from step 1 are mixed together and a long (approx. 2500 bp) deletion cassette is synthesized.
3. To get larger amount of deletion cassette, product from step 2 is amplified, using the same primer pair as in the step 2.
You can find scheme of reaction in attached file.
My problem occurs during step 3. The PCR reaction (conditions, primers, mixes, etc.) is identical as in step 2. After PCR reaction, I find a lot of precipitate in PCR tubes. When I try to analyse reaction in agarose gels, all DNA stays in wells. I've tried to optimize step 3 PCR reaction and different DNA purification procedures after step 2 PCR. I've also tried to synthesize deletion cassettes for different genes. Unfortunately, nothing worked so far...
Do you have any explanation for this effect?
Hi there,
If step2 is working OK, I can't imagine step3, which is basically the extension reaction of step2, not working properly.
That's why I've asked the question :)
In step 2 I usually get few bands in agarose gels (together with a band of a proper size). As a template in step 3 PCR I've tried using:
1. Reaction mixture after step 2 PCR
2. Purified reaction mixture after step 2 PCR (clean-up)
3. Purified band of a proper size from step 2 PCR (gel-out)
Results were the same in all cases.
Hi again,
as a control you could check that the gel purified product from step2 can be used as a template for the different sets of primers used in step1 as well as other combinations like for instance Ftet+Rdown, Fup and Rtet. It would assure it is actually containing the expected sequences with the expected arrangement of fragments. If everything is positive then there is a specific problem with Fup+Rdown reaction.
As a precaution, sequence your product of first step and see if it is what you were expecting.
Hi,
Guess this Q&A will be of some help to you.
https://www.researchgate.net/post/White_precipitate_in_PCR_product-any_thoughts
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