I have been tring to clone a 25 bp(5`-CACCGXXXX....-3` and 5`-AAACXXXX...C-3`) sgRNA into Px458.
I have been using this method : chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://www.csr-mgh.org/wp-content/uploads/2017/04/CRISPR_Cas9_PX458_protocol-1.pdf
However, I am not using plasmid safe
but so far all the colonies that I sequenced (with U6) have no sgRNA in them. I tried to picked 3,6, and 10 colonies but all of which where empty. has anyone faced such a problem? what should be done? any recommmendations?
There is absolutely no need to conduct PCR for the amplification of the gRNA. Simply anneal gRNA oligos with appropriate overhangs, mix with restricted plasmid and ligate. So the first step in the above mentioned protocol shall be skipped. In the second step Golden Gate cloning is suggested. This is also not necessary. Simple ligation for 30 or so minutes at room temperature would be enough.
So the protocol shall look like this: (1) restrict plasmid (check the result); (2) anneal oligos; (3) ligate, (4) transform.
Just have the entire gRNA expression vector made from scratch by a synthesis company. They are so short & cheap, it's unreasonable to try to clone it yourself.
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