12 replies to this topic

[mahrak]

hi there all

I have cloned a fragment in a modified pUC18 vector. there is a XhoI recognition site right at the beginning of the vector( sequencing result has confirmed that.) I have tried digesting the recombinant vector with XhoI(( fermentas, using both fast and regular 10x buffer in different sets of digestion) it didn't work, I used the JM110 ecoli strain which is deficient for dam/dcm methylation system, but the digestion with XhoI still won't work. Has any one came across the similar problem?
any hint or idea is appreciated.

all the best
mahrak

[Curtis]

How do you extract the plasmid?

[phage434]

Can you cut with other enzymes?  What fraction of the digestion volume is your DNA?  Could you give us the exact volumes and components of your digestion?

[mahrak]

I used plasmid extracting kits, Qiagen and fermentas

[mahrak]

I usually digest 1ug of plasmid DNA( 250-300 ng per ul) in a total volume of 20 uls.

[mahrak]

Because of the design I can not use other restriction enzymes.

[mahrak]

it is worth mentioning that I use DNase/RNase free water to elute the columns when extracting plasmid

[andrea_UNSAM]

Hi, I used XhoI with pRsetA. My reaction was succefull: 1ug plasmid, 3 U XhoI (in buffer 3 from NEB because next I do anoter digestion with  BamHI) in 50 ul final volumen ( I don't remember the volumen of buffer, but was the recomendated for the reaction, I think was 5 ul). Incubated for 3 hs a 37ºC. I do that with double digestion from another vector for mi insert (pJet plus insert), with the same 3U, and this was ok.
Even works when I used plasmid purification without purification kit.

[phage434]

I asked about other enzymes because it would tell you if there is a problem with the DNA, making it impossible to cut, rather than that you could use the cut fragment.

So, your typical reaction would have 4 ul of your DNA (250 ng/ul) in a 20 ul volume?  That probably is ok, although I would do this digestion in a 50 ul volume, just to be sure that any inhibitors in the DNA were diluted by the additional water.

[almost a doctor]

In addition to Phage's comment on digesting with other enzymes.  Have you been able to digest a different template with this particular batch of XhoI ? I  know RE are stable and that, but maybe your enzyme is not working.

Also, what's the composition of your reaction (not just the DNA), what's your buffer concentration?  how much enzyme do you add?  And how long and what temperature you digest at?



edit: spelling mistake

Edited by almost a doctor, 19 September 2011 - 07:20 AM.

[mahrak]

here is the composition i usually set up:
1- 0.5-1 ug of plasmid DNA   5ul
2- 10X restriction enzyme  2ul
3- XhoI 1ul
4- D.W  12ul
Incubation: 60-90 min at 37 centigrade degrees
I have tried other restriction enzymes and they work properly, so, i assume the DNA is okie, I have asked colleges of mine to use the same batch of XhoI I used, it works on their fragments, then the enzyme is okie, i suppose.

[phage434]

I would say that either the cut site you are trying to cut is not there, or that it is methylated, preventing it from being cut.  But XhoI is not sensitive to the normal E. coli methylases.  This leads me to believe your cut site is simply missing.  Have you sequenced the region?

[mahrak]

to my surprise XhoI recognition site was exactly there in sequencing results and even using JM110, Ecoli strain with no dam/dcm system, it was still impossible to  cut the site. pretty strange!