Dear Dr. Giles,
I came across your answer regarding how to join 2 pieces of PCR
products together in a single cycle PCR. Please advice me if this is
the right way to produce a single piece of DNA fragment from two PCR
products. Here is my experiment:
1. From cDNA, I have generated two PCR products using two different
primer sets. One product has a size of 500bp (A), while another one is
600bp (B). A and B has around 10-15 homologous bases.
2. Consequently, I need to generate single PCR product using the
forward primer of A and reverse primer of B, to produce a final
product of approximately 1100bp. I have repeated this stage for many
times but still failed. What I get is just smear. I only performed
normal PCR without the 2 quasi-exponential PCR as what you have
mentioned before.
3. Do I need to perform 2 quasi-exponential PCR for the initial PCR;
to produce A and B? Can you please explain the PCR parameters in
detail? Can I replace it with a normal PCR, i.e. initial denaturation
(98C, 2min), followed by 35 cycles of (98, 30sec-> 55C, 30sec-> 72C,
30sec) and finally final extension at 72C, 8min.
4. With the initial PCR product, do you recommend me to use it at a
small quantity (how much would be sufficient?) to perform a single
cycle PCR without adding the primers at high Tm? Does that only
include these steps: 98C, 30sec-> 70C, 30sec and 72C, 30sec, for one
cycle? Do I need to add DNA polymerase at this step?
5. Do I then use this single cycle PCR product from above as a
template for exponential PCR or just add in appropriate primers and
run 15-cycles exponential PCR for this product at primers Tm? Can you
explain the exponential PCR here in details?
Thank you so much for your help!!
Cheers,
Katherine
Kolling Institute of Medical Research
Royal North Shore Hospital
St Leonards, NSW, Australia, 2065
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