I am working on setting up a CRISPR/Cas9 experiment. I have successfully cloned my sgRNAs, but due to the size of my deletion, will need to use a vector template instead of an ultramer. The postdoc in my lab successfully generated pBluescript II KS+ containing his ~1kb gBlock fragment. When following the same protocol, I get ~100 colonies post transformation into DH5a's. After a Qiagen mini-prep and subsequent digestion, I see one of two things:
A) A band the size of my insert, and a band 700bp less than the plasmid - both quite faint, or
B) A band the size of my insert, and a band the size of my vector - both very faint.
When I tried to sequence the colonies from situation A, neither the T3 or T7 primers could work. I am still waiting on sequencing for situation B. I am just wondering what would cause the loss of bp seen in situation A? And why does is seem like so little DNA is present, when the bacterial pellets are of a good size? I have used this kit recently on other experiments and seen very high quantities of DNA.
How much DNA are you putting in your restriction digest? How much in your analytical gel?
The above questions are important. Low amounts of DNA sound like it's not pBS.
T3 and T7 give no reaction or mixed read? If no reaction, you have some other plasmid there instead of a pBS. In my work I cloned gBlocks by adding A overhangs to them, and then cloning into a T/A cloning vector with Blue/White screening, but other approaches should work as well.
Hi
I think you have to repeat your cloning and before going to miniprep try to a PCR screening using T3 and T7 or T3 with one or the other primer you used to amplify your insert.
Do not west your time with what you have it look like a contamination to me.
Bonne chance
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