I set up the digestion as per GeCKO protocol:
5ug- lentiCRISPRv2
3ul- FastDigest Esp3I (thermoscientific)
3 ul- FastAP (thermoscientific)
6 ul- 10X Fastdigest green buffer
0.6ul- 100mM DTT (freshly prepared)
made up the volume to 60 ul with water
digested for 30 min at 37 deg C
Followed by digestion, I ran the entire mixture on a 1% agarose gel and I found a highly concentrated smear in the range of 2-1kb but I didn't find any larger band.
I checked the size of plentiCRISPRv2 by running on the gel after single cut digestion and I found the plasmid was of the right size
Could anyone tell me what could be the reason for this smear?
Dear Saptha,
here are some general problems which might occur: https://www.researchgate.net/post/Smearing_in_agarose_gel_of_PCR_product2
In your case I would first give it a try with a 1,5 % gel, fresh TAE Buffer and running it on lower voltage (e.g. 60 - 90V) and of course long enough.
Good luck,
Hendrik
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