I having some trouble performing a restriction digest with BstBI and FseI on a plasmid. In attachment you can see the gel of the digest I ran:
lane 0: Mass Ruler
lane 1: uncut vector (about 500 ng)
lane 2: uncut vector (about 500 ng) at 37°C, 2h
lane 3: uncut vector (about 500 ng) with 1x BSA and 1x NEB buffer4, at 37°C, 2h
lane 4: vector (about 500 ng) cut with FseI (2 units), 1x BSA and 1 x NEB buffer4 at 37°C, 2h
lane 5: vector cut (about 500 ng) with BstBI (1 unit), 1 x NEB buffer4 at 65°C, 2h
lane 6: Mass Ruler
So, apparently when I add BSA and NEB buffer 4...my plasmid completely degrades! Both NEB buffer 4 and BSA were new and never used before. I also purified my plasmid using a High pure PCR purification kit of Roche to avoid contamination of DNAses...
Anyone can help me out...(My last option is to heat-schock my plasmid again and prep it very carefully)
Edited by tompoes, 17 February 2012 - 09:59 AM.