plasmid can't be digested by enzyme
Posted 24 April 2002 - 01:25 AM
I feel very upset and please you help me!
Posted 24 April 2002 - 03:18 AM
Firstly is the buffer used for double digestion compatable for BOTH restriction enzymes ?
Alternatively, XbaI is sensitive to overlapping dam methylation. If plasmid has been isolated from a E.coli host strain which methylates the plasmid then XbaI will not digest it. If this is the case plasmid should be purified from a E. coli dam strain such as JM110 to get around this problem
Posted 24 April 2002 - 04:42 AM
i use 0.5x k universal buffer that takara recommed. i think it's no problom, but bamH1 in that buffer probably has star activity, and xba1 has not 8o% activity, what can i do about this? i don't know how to purified from a E.colli dam strain such as JM101?
Posted 25 April 2002 - 07:29 AM
Posted 23 May 2002 - 06:35 AM
Regarding the star activity try to use less bamh1 (0.2-0.3 ul).
hope this helps and good luck.
Posted 11 August 2004 - 08:12 AM
Posted 11 August 2004 - 10:48 AM
First off, for anyone who does molecular biology, the NEB catalogue, believe it or not, is a bible. It has tables and charts with answers to questions you never thought of asking but should have.
So, first thing I see is that XbaI and NheI have compatible cohesive ends, so they will ligate together but they cannot be cleaved back apart by either enzyme. I can't tell exactly what you are doing but that might explain your ligation but uncleavability in one of your constructs.
The only one of your enzymes blocked by Dam is XbaI, but ONLY if the restriction site is 5'- tctagaTC -3' in EITHER direction. The small letters denote the recognition site, the caps represent non-recognition site nucleotides directly adjacent which form a Dam methylation site.
Hope some of that helps.
Edited by wirly, 11 August 2004 - 01:03 PM.
Posted 12 August 2004 - 02:08 AM
Great post. I was wondering, why don't you simply cut with one enzyme, qiagen cleanup, cut with 2nd enzyme, cleanup and ligate?
Ofcourse, this way you don't know if the 2nd enzyme has cut. Way I do it:
- split DNA in 2 tubes and digest each with one of the two enzymes
- check both digestions on a gel
- pool both tubes together and add a little bit of each enzyme
- cleanup and ligate
Posted 12 August 2004 - 06:27 AM
If possible (means: available) you could use a vector that already has an insert between your two restriction sites which is big enough to clearly see the difference in a gel between a single and double cut vector.
Ofcourse, this way you don't know if the 2nd enzyme has cut.
I do it like this if I have to mutagenise a fragment and ligate it back into the same vector.
Posted 13 August 2004 - 02:19 AM