I am attempting to make sgRNA but I am having problems with cloning my oligonucleotides into the BsaI-cut pDR274 vector. The problem is that I am left with many colonies containing the empty pdR274 vector, none of which contain my oligo insert. My negative controls (Ctrl 1:. oligos omitted; Ctrl2: oligos + ligase omitted) have a similar number of colonies to my control.
I have made several adjustments to the cloning protocol in an attempt to resolve this problem, but to no avail. These including: 1) changing the duration of vector digestion (30 min, 2hrs, 12 hrs, 24 hrs) and ligation (2 hrs, overnight at 4°C); 2) starting fresh from a different aliquot of empty vector; and 3) dephosphorylating the vector and phosphorylating the oligonucleotide insert. Unfortunately I am still growing up colonies containing the empty pDR274 vector.
Does anyone have any suggestions?
Cheers,
Mike.
This sounds to me that you are transforming with the left over undigested vector from the restriction digest (it does not matter how long you digest you will most likely still have residual amounts of the circular plasmid). You need to gel purify your digested vector after the digestion to remove the residual undigested plasmid then carry out the ligation.
Good luck!
Hanna
Thank you for your replies. Gel purification made no difference because I now know that the problem lied in the oligos. I ordered new oligos and it worked first time.
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