6 replies to this topic

[zhgljj1998]

Did anyone use BamH1 and Xho1 double digestion?

i used    pet28a as DNA 36 (micro l)
              BamH1               2
              Xho1                  2
             10xBSA               5
         10x BamH1 Buffer   5
         TOTAL                     50       waterbath 37 degree 4 hour, gel extraction

     T4ligase   4 degree 2 O/N, found self-ligation?

can anyone help me?

[jadefalcon]

I would always include a dephosphorylation step, even if incompatible ends should in theory not religate. there's another thread around dealing with that problem. I personally found that shripm AP works better in my hands that does CIAP.

mike

edit: check out this thread

http://www.protocol-...?showtopic=4947

Edited by jadefalcon, 27 January 2005 - 07:38 AM.

[badcell]

Have you done separate digestions with both REs to see if both of them are working in the conditions you use? I usually do scaled-down versions of my double digestion (10 microliters instead of 50, f.i.) but using only one of the REs, for the same time and with the same buffer, as a control. That's especially useful when the buffer is not the optimal one for one of the enzymes.

Edited by badcell, 27 January 2005 - 09:10 AM.

[zhgljj1998]

badcell, on Jan 27 2005, 10:09 AM, said:

Have you done separate digestions with both REs to see if both of them are working in the conditions you use? I usually do scaled-down versions of my double digestion (10 microliters instead of 50, f.i.) but using only one of the REs, for the same time and with the same buffer, as a control. That's especially useful when the buffer is not the optimal one for one of the enzymes.

i am trying digestion one by one at the moment. (BamH1 first, then, Xho1). i am going to try total 10 microliter volume of double digestion tomorrow as you suggested. but i am not sure the control is quite useful since there are only 40 bp between BamH1 and Xho1. i may not see any difference between double digested sample and control. do i really need to run 4% gel to see the 40bp, then make sure i get double digested vector? really thank you, man.

[badcell]

You won't see size difference between the single-digested or double-digested, as you said, but you will be able to see if both enzymes are able to cut the vector in the conditions you use for the double digestion. If the BamHI buffer is not optimal for XhoI digestion, then maybe in the 4h incubation, XhoI is not able to digest, so in the control single digestion with XhoI using BamHI buffer you will see lots of supercoiled DNA and just a bit of linearized plasmid- this will mean that most of your plasmid in the double-digest has been in fact digested only with BamHI. Then maybe you need to add more XhoI, increase the incubation time, or do sequential digestions. Hope it helps!

[zhgljj1998]

badcell, on Jan 27 2005, 07:18 PM, said:

You won't see size difference between the single-digested or double-digested, as you said, but you will be able to see if both enzymes are able to cut the vector in the conditions you use for the double digestion. If the BamHI buffer is not optimal for XhoI digestion, then maybe in the 4h incubation, XhoI is not able to digest, so in the control single digestion with XhoI using BamHI buffer you will see lots of supercoiled DNA and just a bit of linearized plasmid- this will mean that most of your plasmid in the double-digest has been in fact digested only with BamHI. Then maybe you need to add more XhoI, increase the incubation time, or do sequential digestions. Hope it helps!

Thank you very much,  Dr Badcell , try what you suggested. finally, finally, get right sequence result back. my problem is fixed. anyway, i digested one by one, and incubate more than 10 hr in 37 degree water bath either.  

[badcell]

That's good!
I'm glad it worked, zhgljj1998