My product size would be around 5kb with 5' and 3' flanking regions. I have good DNA and primers also worked in other experiments, I was wondering how should I optimize the PCR conditions. Any suggestions?
Hi,
This is on the large side. So you would need to use some proofreading polymerase and increase the elongation time to a minute per Mb. I think 30 cycles should work. Some stretches are still hard to amplify though. After you clone it (I am guessing it is for cloning) make sure to sequence the insert. Depending on the vector you might be able to put the gene and the promoter separately. Hope that helps!
Thank you, Svetlana, that helps.
Actually yes, increasing the extension time and adding DMSO worked for me. I am going to sequence the insert.
Cool! Cloning can be really tricky. Everyone in the field struggles with it at least once...
I suppose first you should need a high fidelity polymerase capable of proofreading and having long processivity.
- Badges
- Science method