I am trying to clone my target sequence for CRISPR/Cas9 work. I have performed digestion and gel purificatoon of the lentiCRISPRv2 plasmid with a good yield and purity. However, when I performed my ligation with the annealed sgRNA oligos (1:200 dilution), I cannot get any colonies for my sgRNAs whereas I can get a good colony count for my +ve control bacterial transformation using DH5a. My insert oligos will be as follows:
forward sgRNA: 5' CACCGAATCCCGGCGTGTCCACGA 3'
reversed sgRNA: 5' AAACTCGTGGACACGCCGGGATTC 3'
insert size: 24 bp
vector size: 12kbp
I have followed all protocols from Zhang Lab but still didn't able to get any colonies.
I tried to do ligation using Quick Ligase kit (NEB) and T4 DNA Ligase (Promega) but still no results.
Which part should I troubleshoot so I can get result that I want?
What restriction enzyme are you using? BbsI? Usually to maximize U6 promoter activity we design sgRNAs with an extra G. thus it would be:
forward sgRNA: 5' CACCgGAATCCCGGCGTGTCCACGA 3'
reversed sgRNA: 5' AAACTCGTGGACACGCCGGGATTCc 3'
I never dilute the annealed oligos that much, usually use 1uL of a 5uM anneal reaction. Maybe around 600nM in the ligation reaction.
Hope this helps,
Best.
Hi Diogo Figueiredo . Thank you for the suggestions. Yes, we will add extra G for the sgRNAs. For the dilution of annealed oligos, I followed from Zhang's protocol. Do you have any supporting documents for your protocol? Thanks in advance!
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