Currently, I am using a plasmid containing two gRNA cloning site. The restriction enzymes used for cloning gRNAs into this plasmid, pX333, are BsaI and BbsI. I digested the pX333 plasmid using BbsI restriction enzyme and performed ligation with the first gRNA. I submitted the plasmid for sequencing and found that the original filler sequence for the BsaI cloning area was now positioned in the region where the BsbI sequence was removed after digestion. In other words the gRNA sequence was not inserted but instead the BsaI filler sequence (sequence between the two BsaI cut sites) was inserted. I am unsure as to why this occurred. Any suggestions?
Highly interesting - genetic cloaning - recombinant DNA technology .
Maybe you have used a "generic" gRNA sequencing primer? I usually sequence with a U6 primer that in this case might "catch" both sites.
Could you provide the sequencing primer sequence you use?
Moreover, in most cases, before plasmid isolation I pick several colonies and perform colony PCR with the reverse oligo of the gRNA and a U6-F primer. Then I isolate the plasmid only from the positive colonies and validate via sequencing.
In this case, where there are 2 cloning sites, you might want to use different primers for each site
BbsI site:
For - gaatggcgcatgtgagggc
Your gRNA1 reverse oligo
BsaI site:
Your gRNA2 forward oligo
Rev-ccgtaagttatgtaacgggt
Please note that these primers were designed for the matter of this answer and were not tested.
Good luck
Hi Roy,
I designed two different primers to sequence each sgRNA sequence. A forward primer targeting Orf and the a reverse primer targeting CMV. The Orf primer sequences the first gRNA cloning site (U6-sgRNA-gRNA scaffold), while the reverse CMV primer sequences the second gRNA cloning site (gRNA scaffold-sgRNA-U6). I have only performed the BbsI-gRNA cloning as of now and wanted to check that the first gRNA was inserted properly. This plasmid has both BbsI and BsaI cutting sites for inserting two gRNAs.
I do not think the issue are the primers that were used. I am unclear as to why the filler sequence between the BsaI cutting sites was re-positioned to the BbsI cloning site after digestion with BbsI. How can this unwanted recombination occur.
I am sequencing the original plasmid to be sure that the map available on Adgene is accurate.
Just an update - after sequencing the original undigested px333 plasmid the sequence of the two sgRNA cloning sites were correct. So I am still unsure of how the swap occurred when digesting with BbsI.
I repeated the cloning protocol using PlasmidSafe exonuclease to limit unwanted recombination. The plated transformed cells appears to be as expected. I plated cells transformed with BbsI-digested px333 (few colonies), sgRNA1-pX333 (many colonies), or sgRNA2-pX333 (many colonies). In the past cloning experiment, bacterial cells transformed with sgRNA1-px333 or sgRNA-px333 resulted in few colonies, which is what I would expect (based off of past experiences).
Update: I sequenced my plasmids today and the cloning was successful after using the PlasmidSafe exonuclease in the reaction. There was some unwanted recombination with BbsI digestion. If anyone has any questions, do not hesitate to reach out.
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