Based on Fengzhang lab protocol provided on their lab website,I performed the one step digestion and ligation, I got a lot of colonies on negative control plate. I am wondering the cut sequence from the plasmid will be ligated to the plasmid, so why the negative control plate should no colony grows? The pX459 plasmid was used and I did not treat the ligation product with PlasmidSafe exonuclease.
I guess Bbs I does not completely digest your plasmid.
If one of Bbs I site is only digested, the plasmid fragments would be self-ligated.
You could avoid the problem with phosphatase treatments after Bbs I digestion.
Hi Hitoshi,
Thanks for your answer. If you digest plasmid first, you can treat the plasmid with phosphatase to avoid the problem. My question is how to explain the negative control result with single step digestion-ligation? Assume the plasmid was cut completely, the cut fragment still can re-ligated to the plasmid in one step protocol, so still can get colonies on negative-control plate.
I repeated this assay once using new ordered BbsI enzyme, the negative control plate still has tons of colonies. So as I have mentioned above, the one step digestion-ligation protocol can not avoid self-ligation. The good news is I got all correct insertion this time.
Note: 1.BbsI is very important for the successful cloning. The BbsI activity will lose after long time storage in -20degree and they (NEB)recommend to store this enzyme in -80degree.
2. I also extended the digestion-ligation cycles to 30, then all my 7 constructions are 100% correct and 4 colonies were picked for each transformation. The insertion was verified by double digestion with NdeI and BbsI (as showed in attached photo, the last lane of each row is the empty vector control) and also verified by sequencing.
3. The following link is a very detail CRISPR cloning protocol and it is very useful.
https://benchling.com/protocols/5DmqRd/crispr-mediated-gene-disruption-in-ch12f3-2-cells/sbs
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