Hello all,
I am working with PLJR965 CRISPR plasmid size 8.6kbp. I extracted the plasmid by Alkaline Lysis Method and got 4 bands. Now I am confused that if I have extracted the plasmid successfully or no? and If the other bands are isoforms of this plasmid why there is no band near 8 kbp.
Other details:
1. 0.7 and 0.8% Agarose
2. Voltage: 80V
3. Ladder 250bp-10kbp
Uncut plasmids run as a supercoiled and an open circular version. Both tend to run smaller than the linearized plasmid and are hard to size accurately. You really only have two bands, the very top may be something else. Best thing is to cut your DNAs with a restriction enzyme that gives a characteristic pattern that you can size accurately.
I agree with Gregory Dressler , there look to be 2 plasmid bands (or a smear). The upper band might be gDNA. I agree that trying a few different digests would help. Good luck!
Hi Amy,
It seems four bands after alkaline lysis of PLJR965 CRISPR plasmid is normal, representing different topological forms of the plasmid. Absence of a band near 8.6 kbp suggests low amount of supercoiled plasmid DNA. To obtain supercoiled DNA, purify the plasmid using a gel extraction kit or optimize alkaline lysis protocol.
If you have any further questions you may write back!!
Best of luck!
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