Cloning Problem: HSP90AA1 - Cloning Problem: HSP90AA1 (Feb/22/2011 )
I am cloning Homo sapiens heat shock protein 90kDa alpha (cytosolic), class A member 1 (HSP90AA1).
But i am not able to amplify it.
I think primers sets are not perfect. I want to know whether Overhang (Restriction sites and other nuclotides) should be counted while calculating Tm of the primer for cloning.
If the primers are correct then what are the possible ways to get rid out of it.
I came to know that this gene is difficult to clone. if it is so then what should i do.
Is anybody working on the same gene? if so please HELP!
The non-specific parts (restriction sites, kozak sequence, spacers, etc.)of the primers should not be included in the Tm calculation. Have you tried adding secondary structure inhibitors such as DMSO?