Hi,
I'm currently trying to generate a one-vector plasmid system for a knockout using CRISPR/Cas9. I'm using lentiCRISPRv2 (ampicillin-resistant) and following the GeCKO Target Guide Sequence Cloning Protocol from the Zhang Lab. So far, I have been unable to get any colonies to form on my plates treated with plasmids that have theoretically been re-ligated with my target sequences inserted. I've had no issues getting colonies on plates treated with undigested plasmids, suggesting that neither the bacteria (Stbl3) nor the plates are my issue. So the other steps that could potentially be where something is going wrong are:
1. Restriction Digestion
I have digested lentiCRISPRv2 with Esp3I (Thermo) and BsmBI-v2 (NEB) on several occasions. Both digestions seem good, but I will say the BsmBI-v2 digestion looks cleaner. After digestion, I gel extracted the larger molecular weight bands, leaving the 2kb filler behind. I used NEB Monarch Gel Extraction Kit for this, using the supplied elution buffer to elute the DNA after extraction. The product being elutes is ~12kb, so I have followed the guidelines in the protocol for products >8kb. Upon NanoDrop, yield is fairly low, but 260/280 ratios are normal and concentrations are within a usable range. The 260/230 are a little low, but I think this is due to the large volume of dissolution buffer needed for my cuts and the amount of guanidine salts present.
2. Oligo Annealing
I have checked all of my oligo sequences and their overhangs, and all seem to be correct. I am using T4 PNK (NEB) and T4 DNA Ligase buffer (NEB), as the buffer supplied with the PNK does not include ATP. I'm not sure how to verify the success of this step prior to proceeding, so I feel I have to trust the process has worked until I reach bacterial transformation. Is there a way to verify the annealing of the oligos? I am currently thinking I could use EtBr and run my annealed oligo samples alongside non-annealed oligo samples on an high% agarose gel (products are only 20 bp), considering that EtBr has a higher affinity for dsDNA. Is there anyway for me to assess whether my oligos could be annealing improperly, preventing their insertion into the plasmid?
3. Ligation of digested plasmid and oligo duplex.
I am currently using Quick Ligase Kit from NEB for this step. '
Does anyone have any thoughts on high I can perform any verification of these steps or any potential complications I have not considered yet?
Thanks for the help!
Seth
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