Problem with BamHI Digestion - (Dec/14/2014 )
Hello,
I am a student in a Biotechnology program and as part of my Mol Bio class, I am attempting to identify an unknown plasmid as one of 10 on a list. I looked up all the information, and each is listed as having at least one BamHI site. However, when I run a digest using it, BamHI does not cut at all.
Conc. DNA: 43 ug/ul
Digests tried:
1 ul DNA
1 ul Buffer E
7 ul Nuclease Free Water
1 ul BamHI
Digest in water bath @ 37C for 1 hr
also
2 ul DNA
1 ul Buffer E
6 ul Nuclease Free Water
1 ul BamHI
Digest in water bath @ 37C for 2 hr
Add 4 ul loading dye
Load 10ul to well
run ~1 hr until dye is ~1cm from end of gel
I am trying to write up my results, but can't figure out why BamHI didn't cut. Any suggestions?
Thanks,
Jeannine
Your DNA is really 43 micrograms per microlitre? That is an enormous amount...
Too much DNA and you won't see the cut DNA. Usually restrictions are done with at least a two-fold excess of enzyme over DNA.
Sorry wrong units, re-checked my notebook, the DNA concentration is 43ng/uL.
Ok, that makes more sense. Do other digests work? What does the uncut look like?
Attached is a word document with an image of the three gels I ran. In the first two gels, the bands in the lane BamHI was in matches the bands in the uncut lane. In the third gel, HindIII did not cut by itself (bands matched uncut lane) but there are 4 bands in the lane with BamHI/HindIII. EcoRI only cuts once in the first two, but there are four bands in the lane that contained EcoRI/HindIII on the third gel. Edited to add - Also, sorry, I just saw the student section where this should have been posted.
Gel 1: BamHI, EcoRI, & HindIII
Lane 1: empty
Lane 2: Lambda ladder
Lane 3: uncut plasmid
Lane 4: BamHI digest
Lane 5: EcoRI digest
Lane 6:HindIII digest
Lane 7 & 8: empty
Gel 2: EcoRI & BamHI digest
Lane 1 & 2: empty
Lane 3: Lambda ladder
Lane 4: uncut plasmid
Lane 5: EcoRI digest
Lane 6: BamHI digest
Lane 7 & 8: empty
Gel 3: HindIII, BamHI/HindIII, EcoRI/HindIII
Lane 1: empty
Lane 2: Lambda ladder
Lane 3: uncut plasmid
Lane 4: HindIII digest
Lane 5: BamHI/HindIII digest
Lane 6: EcoRI/HindIII digest
Lane 7 & 8: empty
Ok, looks like your BamHI is dead, it is probably the enzyme rather than anything else. Did you add BSA (can't remember if E comes with it included)?
I did not add BSA and it looks like E does not contain it. Others used BamHI from the same lot and had no problem, so I thought that would have ruled out enzyme inactivity. The enzyme was aliquoted into 1.5mL microfuge tubes. Is it possible for one tube to become inactive while the others are ok? Would multiple freeze-thaw cycles cause that to happen? The tubes were stored at -20C, thawed for use, then re-frozen until used again. Thanks for all your input.
From this I take it that while buffer E does not contain BSA, the enzyme itself does: https://www.promega.com/~/media/files/resources/technical%20references/restriction%20enzyme%201x%20buffer%20composition.pdf
(If the enzyme was supplied by the same company)
So this should not be an issue.
Is it possible for one tube to become inactive while the others are ok? Would multiple freeze-thaw cycles cause that to happen?
Enzymes are usually in (50% or so) glycerol, hence they do not freeze when at -20°C and you do not need to actually thaw them before use. Having an enzyme at RT for a prolonged period of time will decrease its quality significantly. You should always keep enzymes on ice or in a freezing block, take them out only immediately before you need them and put them back into the freezer immediately after.
Perhaps the aliquot that you used has been "forgotten" at RT too long and thus does not work anymore. Especially in a lab course, such things can easily happen by accident because many different people work with the same substances, perhaps for the first time - at least during my courses, this was the case a few times :D