I am trying to clone 3kb gene into pGEM-Teasy . To amplify the insert , i am using pfu , so i do poly(A) tailing. I am taking 135ng/ul of amplified gene to add poly(A) tailing. After that , i am taking 1: 3 vector to insert ratio. Inspite of all this , i am getting more blue colonies with little number of white false colonies. pls help me in cloning
Clear it what you want to ask....i am unable to get you.......Pfu will be used after you join the primers to your gene....so i think you should first choose primer to your gene then poly a tail addition and all will only work
Actualy i am using pfu for error free amplification of my gene and as we know it does not add poly(A) at the end of amplified product as added by Taq. So i am doing poly(A) tailing before doing ligation into pGEM-Teasy vector. I am not inserting my gene in MCS .
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