Hello,
I am making some plasmid constructs via Inverse PCR and T4 enzyme ligation.
I started with a plasmid that housed nucleotide sequences for two fused proteins. My goal was to cut out the nucleotide sequence for just one of the proteins.
For my Inverse PCR primers, I designed the forward primer to be at the beginning of the desired protein. The primer was 35 nucleotide long
I designed the reverse primer to be 34 nucleotides long. It included the vector sequence just before the undesired protein.
After amplification with inverse PCR, I ran the sample on the gel and if the band was correct, I purified the DNA from the gel.
I then added I 2ul ligase buffer and 1ul T4 PNK and incubated it at 37C for 30 minutes. Then I let it sit at room temp for 5 minutes before I added 1 ul ligase.
Then I left it at room temperature for a few hours and before I stored it in 4C overnight.
I have done this process before and it has worked for me. However, I keep running into an issue where I continually have a one nucleotide deletion right at the site of ligation.
For example,
If I want this sequence: ATGCAC
I will get this sequence instead: ATCAC
That "G" is right at the junction. For some reason, it continually gets deleted during ligation.
I was wondering if anyone has any suggestions on how to prevent this from happening? Maybe there is something wrong with the procedure that I am using to ligate?
There’s too much passages pcr unless is use the most precise polymerase it will in errors. It’s better to order a gene with the enzyme cut adaptors included , and you’ve just to verfy the out of frame mistakes before ordering using your software.
Gel purification can sometimes lead to DNA damage or loss, which may contribute to mutations during ligation. Consider alternative purification methods like column-based purification kits to minimize potential issues.
If your primers are not purified by gel electrophoresis or chromatography, then there is always a chance of losing one letter (or maybe two) from the end of the primer, and then after ligation of the PCR product from such primers, these letters will be missing at the junction.
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