I have been trying to digest a vector pXMCS for the last two months but no luck so far. I have been using NdeI and KpnI (Both of them were present on the plasmid map of the vector) as restriction enzymes and incubating the reaction at 37 degrees for 5 to 6 hours. I am using buffer 2.1 which is compatible with both enzymes. Even overnight digestion does not seem to work out. I also tried single enzyme digestion reactions for 5 to 6 hours and overnight (just to check which one of the enzymes is not working) but this is what I got (2nd picture). I would love to get some suggestions on the same. Thank you.
PICTURE 1: Lane 1: 1 kb plus ladder, Lane 2: uncut vector, Lane 3 and 4: Digestion reactions (with 1ug concentration vector each), Lane 5: Uncut vector, Lane 6: Digestion reaction (3ug vector)
PICTURE 2: Lane 1: 1 kb plus ladder, Lane 2: uncut vector, Lane 3: KpnI, Lane 4: NdeI
When were the enzymes purchased and how many freeze/thaw cycles have they undergone? Were they immediately put back in the freezer after use by all users?
It seems to me that you reversed the image numbers? But, if I put the legends back on the correct image, I have the impression that your digestions worked well. The enzymes work, because your vector seems to have been linearized, compared to your undigested control which includes the plasmid in different circular forms (- coiled, super-coiled, etc.) which have "aberrant" migrations in agarose gel compared to linear DNA.
What you would need to tell us next is the number of restriction sites you expect, and the expected sizes of any plasmid fragments.
Otherwise, in addition to what Robert suggests :
By what technique was your pXMCS vector purified? Would there not be digestion inhibitors left? (salts, phenol, EtOH, etc.)?
Have you ever tried to re-purify your vector?
Good luck !
@Robert Adolf Brinzer - Purchased it 2 years ago I think. Not too sure about the number of freeze thaw cycles. Yes, they were put back in the freezer immediately after use. Both enzymes seem to linearize the vector individually but I dont see the second band after digestion in the presence of both vectors.
@David Lepetit - i am expecting two bands. One around 3kb and the second band between 700bp to 1kb. The vector was isolated using a kit (gbiosciences)
I tried purifying it several times with a different kit as well but I am getting the same result. Should I try isolating the vector manually without the kit?
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