I want to clone a gene into pET-15b using overlap extension PCR. The problem is that the melting temperature of the pair of primers is 82ºC and 91ºC afor Q5 polymerase and I think that I don't have another place to clone the gene. Is it possible to have amplification with these temperatures? Thank you.
the fact that your annealing temperature is 82 ( for instance) means that it will anneal at all temperatures below 82 at efficiencies of over 50% so any lower temperature will work in a pcr. The problem with low annealing temps is usually that the primer binds to the wrong places in a large genome but this is not the case here. You have a very clean reaction mix with little dna other than the proper target so just do a 2 step pcr at the temperature the enzyme works best at....eg 94c then 72c if the enzyme works well at 72 0r 94c and 68c if 68 is the right enzyme temperature
I think you can try to do with gradient Ta to screen the optimal Ta for your PCR, firstly. On the other one, you can use either Infusion or Gibson for your other solution. if target gene that you want to clone into plasmid is shorter 2000bp in length, I would like to suggest you that pJET is a worthy of consideration. Actually, it is important to consider hairpin during overlaping so you should check the tempurature which makes hairpin structure to avoid. For example, if hairpin is at 70oC, you should choose the temperature at 75oC or higher. Temperature annealing should be higher 5oC than Tm hairpin. Hope to help for you!
Try optimization at 82-85c,as it may help achieve desired results. Good luck
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