I am using Stratagene Quikchange SDM kit and protocol to mutate my gene of interest which is inserted into pET151D TOPO from invitrogen. I got the sequencing result and the gene is mutated. However, there is an extra 82bp in the middle of my gene near the mutation site. I checked the sequence of that 82bp and it resembles the primer sequences that I used. My primers are 29bp long. In that 82bp, i can see several segments that matches exactly with my primers. I am already using an annealing temperature of 62C. Anyone know how to solve this problem?
Thx
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#3
saltycookie
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Posted 20 February 2009 - 03:30 AM
Do you have only one colony? Try other colonies. You will get a right clone. I guess the additional 82bp insertion may result from your mutagenic primers. Both primers may not only perfectly match but also partially match each other. The partial match makes your insertion bigger.
#4
scolix
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Posted 21 February 2009 - 02:22 AM
saltycookie, on Feb 20 2009, 12:30 PM, said:
Do you have only one colony? Try other colonies. You will get a right clone. I guess the additional 82bp insertion may result from your mutagenic primers. Both primers may not only perfectly match but also partially match each other. The partial match makes your insertion bigger.
have you sequenced the original vector to verify if this mutation is present. Also sequence other colonies, hopefully they are fine.
