Nanodrop question - Spectrophotometrical measurment of bisulfite treated DNA (Nov/30/2006 )

Thanks Nick.

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Hi Branco,

sorry, I just saw that I have written 65°C as annealing temperature - that surely was wrong, I ment 65°C as elongation temperature - annealing should be like Nick has said. Sorry for that...


Nick, I generally agree with you on primer design, but in this case, I use the same primers and have never seen a C outside a CpG in the sequence... This should indicate full conversion, shouldn't it?

Krümel

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Hi Krümel,

Should I set elongation temperature to 65 C just for 2 round of PCR (nested PCR)? How about first round? Should it be 72 C?

Branco


PS (to moderator): I apologise because discussion has gone outside of initial topic (nanodrop)

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Hi Branco,

I tried it for both rounds, but it seemed necessary only for the second round? Doesn't make too much sense, I know, but it works well for me. For the reelin primer; I use 55°C as annealing and 72°C as elongation for the first round PCR, cycle conditions following Nick's protocol. The second round is slightly different due to the modified primers.

Krümel

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Thanks for the tips Krümel.


Branco

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@ Krümel.

if you are seeing full conversion with your primers then it should be okay. I wouldn't change anything.

@branco.

I am pretty relaxed here, doesn't matter to me if you go off topic.

Nick.

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