after miniprep, I sequenced the plasmid. After sequencing I found that a region of plasmid was missing and its size if of around 8kb while it should be of around 12kb. It has happened to me for the second time. Kindly suggest measures to tackle this.
You have add more information about the numbers your "missing region" and the enzymes you used. expected size of lenticrisprv2 is 14,873 bp.
Hi Uwe Borgmeyer
I have used Esp3I (Bsmb1) from thermoscientific. It is supposed to cut at 2234 and 4119 position. But I am getting another 4kb region missing after the 4119 position. after 2234 bp, the plasmid sequence is starting after 8100bp. It has occured a no. of time. kindly suggest me why is this happening and how should i tackle this.
Thanks
Lentiviral plasmids tend to have repetitive sequences which means they can recombine during plasmid prep. Some useful tips to avoid this-:
1. Use bacterial cells which have low rate of recombination. I use stellar chemical competent cells from NEB or Stbl4 and have had good results
2. Incubate plates after transformation at 30 instead of 37. This also helps reduce recombination
3. Incubate only for 12-16h. Chances of recombination increases when you incubate them for longer.
Hope this helps
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