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plasmid can't be digested by enzyme


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#1 rainlily

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Posted 24 April 2002 - 01:25 AM

I use the method  of molecular clone to extract pBI121 plasmid, and digeste it with Xba1 and bamH1, but i found it is not be fully digested, so i did not continue to do  construction of plasmid vector.

I feel very upset and please you help me!

#2 Graeme

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Posted 24 April 2002 - 03:18 AM

Two possible reasons;
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

#3 rainlily

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Posted 24 April 2002 - 04:42 AM

thank you for your reply.

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?

#4 Graeme

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Posted 25 April 2002 - 07:29 AM

If the buffers and enzymes are ok its most likely a problem with dam methylation. If you look at the back of any mol. biol catalogue (eg. stratagene, promega) you will find a list of E.coli strains. What you want is one which contains a mutant dam gene,this will be  listed in the bacterial phenotype. JM110 is one particular strain which exists but others are also available. If you can get one of these strains then prepare competent cells by your usual method then transform your plasmid of interest. Pick and grow up a single colony then miniprep your plasmid. Try digesting this plasmid now and you should find digestion goes to completion.

#5 Asif

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Posted 23 May 2002 - 06:35 AM

How about steric hinderance. When you do double digest, if your digestion sites for the two enzymes are quite close it might be difficult for the two enzymes to bind and thus steric hinderance. Try doing one by one, digest with xba1 first, after 2-3 hrs of digestion add 0.1 volume of 3M sodium acetate (pH5) and precipitate the dna with 2 volumes of ethanol (100% ethanol) --> incubate in - 70C (minus 70) for 1 hr--> spin at max speed for 15 min--> throw supernatant without disturbing the area you expect the pellet to form (most of the time you would not be able to see a pellet) --> wash with 70% ethanol(500ul) --> spin at max speed for 5 min--> throw supernatant (as above) --> dry pellet (your cut dna) --> prepare second enzyme reaction --> 2-3 hr of digestion with bamh1-->run gel to check .

Regarding the star activity try to use less bamh1 (0.2-0.3 ul).

hope this helps and good luck.


#6 Downs

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Posted 11 August 2004 - 08:12 AM

I have a similar problem. I've been working with a bactrerial gene in pCR2.1-Topo and have had no problems inserting it or excisisng it as an Asc1/Pac1 fragment or an Nhe1/Xba fragment. But I've now started working with a plant gene 500bp long, engineered with Asc1/Pac1 and Nhe1/Xba sites by PCR. The problem is I can ligate it into the plasmid ( it shows up on PCR of plasmid preps) but am not able to cut it out. Is this a methylation problem? The strain it is in is Top10 E.coli. Any suggestions would be gladly welcome.

#7 wirly

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Posted 11 August 2004 - 10:48 AM

Downs-
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.


#8 InvisibleSurfer

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Posted 12 August 2004 - 02:08 AM

Asif,

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

#9 CKM

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Posted 12 August 2004 - 06:27 AM

Ofcourse, this way you don't know if the 2nd enzyme has cut.

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.

I do it like this if I have to mutagenise a fragment and ligate it back into the same vector.

Greetings
CKM

#10 InvisibleSurfer

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Posted 13 August 2004 - 02:19 AM

Sure CTM, but in most cases when a gene is cloned into a vector, many of the sites you/we are interested in are missing. But yes, this is a very straightforward way of making sure both enzymes have cut.

#11 benni

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Posted 22 February 2005 - 09:23 AM

E.coli Top10 have dam methylation ?




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