In order to do clone confirmation by digestion,I have to double digest my recombinant vector to find out my desired insert is ligated into my vector.
I have to digest it with NdeI and BamHI. Our digestion condition is:
12 µl vector
4 µl tango buffer (2x)
0.5 µl NdeI
0.5 µl BamHI
DDW 4 µl
Final volume 20 µl
3hrs---37'C
When I digest it I should see a 1200bp and 5800bp bound on agarose gel.But I only see one bound with 7000bp length.(when I digest my gene with one of this 2 enzymes,I must see 7000bp bound and I succesfully saw it)
what shoud i do?I thought maybe one of this enzymes inhibit activity of the other.so first I digested it with BamHI and after 3hrs I inactivated enzyme with 80'C 20min. then I added NdeI and incubate in 37'C.but when I electrophorased it I saw 7000bp bound again.
thank you for helping me
It sounds like you may already have done this, but if not I would recommend that you set up a series of control digestions in identical buffer conditions:
1) DNA only
2) DNA+NdeI
3) DNA+BamHI
4) DNA+NdeI+BamHI
This will allow you to confirm that both enzymes are active under near-identical buffer conditions, which may reveal that one of the two enzymes is not active or that one of your cut sites is missing/mutated.
New England Biolabs' (NEB) materials suggest that NdeI and BamHI (or BamHI-HF) are compatible in several buffers (e.g. Cutsmart at 37 *C).
Did you load your uncut plasmid along with digested plasmid? If so, did you see any difference? This is a very important control. I doubt whether the enzymes worked.
I was about to suggest the same as Bower. It looks like one of your enzyme has not worked. Set up different reactions as Bower explained. Run the gel and look for difference between the coiled undigested and digested linear DNA. If both single digests are looking perfect, then try to ethanol precpitate after first digestion, resuspend and digest with the second one so as to avoid any conflicting ingredient from the previous digestion.
Thank you all for your help
- dear brian,yes.I exactly did what you suggested. both enzymes work in single digestion, as I told before, the digested vector with only NdeI and only BamHI showed up in7000bp. as same as double digestion. In fact this three are seen on exact same bound (7000bp).
- dear ramachandramouli, yes i did it and they are different. I'm shure that my enzymes work in single digestion
- dear beena both single digestion were perfect and I 'm going to do what you suggest
Hi there,
Do you mean that both enzymes used individually are both active and that they can't work together although sites are not contiguous? This sounds very weird to me...
So then
1. you can change the buffers and see
2. or you can do single digestion, run the gel and elute the band. You can do single digestion with the second enzyme. This should work.
It looks strange and wired. I agree with all the suggestions given above. Did you try first digestion with both of the two enzymes? I am wondering if only one of the enzymes inhibit the other one. If you have not done it, you can try digestion with BamHI first followed by inactivation and then digestion with NdeI or vice versa. If this is not work, you will apply Budida's second suggestion above.
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