I was doing site-directed mutagenesis using PCR. And I have ordered forward and reverse primers that are reverse complements of each other (both primers are around 45bp, 7 aa before and after my mutation residue).
After the PCR reaction, I can see a clear band with the correct size in the agarose gel. After DPNI treatment the PCR product is cloned, and then single colonies are picked and sent for sequencing.
However, when I got the sequencing result, although one of the colonies had a perfect result, (I am using pGEX vector so GST sequence + PreScission site + Target sequence + Stop codon)
Most of the other results showed multiple continuous primer sequences (GST + PreScission site + Target sequence but containing multiple times of primer + Stop codon)
[image attached, my primer sequence is highlighted]
I am confused how this could happen.
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