I need to amplify a pcDNA3 plasmid containing human SCN9A CDS insert (pcDNA3-SCN9A construct). The CDS is around 6 kb, and the total construct length is approximately 11.2 kb, which is a bit lengthy. During the initial days of the experiment, the amplification of pcDNA3-SCN9A was OK without any events of recombination. But recently, when I try to amplify the same plasmid, I am getting repeated events of recombination around the same region (verified by RE enzyme digest and sequencing). My primary aim is to do site-directed mutagenesis with that plasmid.
I tried the following:
1. During the start of the experiment, WT plasmid was usually amplified using DH5a at 37 degrees C and the colonies were positive without no event of recombination (verified by RE ezyme digestion & sequencing). However, the colonies would appear after longer incubation time (30-36 h).
2. I did the SDM using a fragment from the pcDNA3-SCN9A construct inserted into the pBluescript SK (+) vector and the SDM was successful. However, when I tried to put the fragment back (RE digest, ligation & transformation) into the original construct (pcDNA3-SCN9A), I started observing recombination events. This is where things started to get complicated and strange.
3. So, to reduce the chance of recombination, I tried transforming the constructs into DH5a, and DH10B competent cells incubated at 30 degrees C. Yet the results are same - recombination still occurred.
4. Now when I try to amplify the original WT (pcDNA3-SCN9A) construct using DH5a and DH10B competent cells at 30 degrees C, strangely the WT plasmid too showed the presence of recombination (verified by RE enzyme digest and sequencing).
I have no idea as to what is going on with my construct. Had anyone encountered similar issues like mine? What would be a better strategy to overcome the recombination event? Should I stick to 37 degrees C or 30 degrees C for incubation?
Please help me out with this issue.
Though DH5a and DH10B are recombinase negative bacteria, several E. coli strains have residual recombination activity by different recA alleles (see article down below). You can check your E. coli for recombinase activity. Hope this might help you.
Article Plasmid Transformation of Escherichia-Coli and Other Bacteria
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