i did cloning for cytochrome P450- 2D6 into pGEX-6P-2 plasmid vector. when i screen using PCR i get the band of PCR product at an actual size of 1375 bp. but when i use double digest i do get double bands but the lower band is above the expected band of 1375 bp. i would like to know what are the causes since when i check my marker, it shows that the gel was run properly?
Mohammed Milhim
That is normal....the restriction enzymes (specially if they are HF) stay bound to the DNA causing a gel shift. If you want to remove the restriction enzyme from the DNA fragment you can add SDS to your DNA loading buffer.
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