Sequential digestion - Sequential digestion using two RE with different incubation temperatures and dif (Oct/03/2005 )

Hi there !

I am doing this sequential digestion for some time without success.
There I am using two restriction enzymes Vsp I and Bcl I having temperatures 37 and 50 degrees respectively.
Well ! the buffers for them are Vsp I - Promega Buffer D , Bcl I - Roche Buffer M.

I have tried to swap the buffers and see the activity and it seems that Bcl works well with Promega buffer D . so I am doing a sequential digestion using this for both of them ie for vsp I and Bcl I. due to incubation temperature difference .

Could anyone please help me out as how to get a satisfactory digestion with this setup.

Thanks !!
Have a nice day

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hi
Bcl I is a dam methylayted site. And this inhibits the activity of the enzyme. You musty produce your plasmid in a dam6 / dcm- strain (for ex Ecoli 110)
fred

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hi thank you so much fred..

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hi ! again..
well I am doing this digestion with mouse gebomic DNA and not plasmid.
also how best can i use this (vspI and Bcl I ) combination to minimise the dam effect as well as maximise the cutting .

Hope this is feasible ...

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Roche19 asked once if commercial demethylases were available. But no replies to his post and i've not found any one which may be to purchase. Well bes possibility would be to pcr amplify a fragment of the genome that interests you and to produce it with a plasmid (like topo kit) in a host strain that do not methylate dam and dcm...

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Thank you for your help fred.
I have actually amplified using PCR that particular segement of DNA where the Bcl I site is situated .. However then I am getting a good digestion using Bcl I from Roche.
I am checking all this digestion steps because I am having this problem to get bands in my southern blotting.
Basically i am using Bcl I and Vsp I to digest mouse DNA and get a 8.9kb fragment which I plan to detect using a 500 bp probe.
This is troublesome and i am not getting any band in the membrane .. hence thought to reflect to the digestion procedure. if u can shed any light on this it could be great.

thank you..

meeta

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hi
you can check your digestion steps in a "standard" agarose gel and see if degradation of your DNA occurs.
After this point, is your labelling (radioactive i suppose) efficient?
what strigent are your washing conditions?
these are i think more critical steps as a restriction step becaus with a 500bp probe, you should see sthg, even if it was a background!
fred

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Meeta,
As far as southern is concern, it is a very sensitive technique. It can even recongise even 1 picogram of DNA. Have you check your SSC,Denhardts, BSA for contamination. There may be some contamination in template DNA. First make these things sure.
Best wishes
amit

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Hi amit
well I am sure about the buffers and the prehybridisation mix which i am using since I am using the ambion prehyb solution.
about the DNA the mice DNA I am getting from another source so i am not sure as how they do the purification and all.

do u think phenol and ethanol ppt can cause problems to my DNA for southern .

thanks again
meeta

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Well phenol and residual ethanol can of course interfere in the digest , can I ask you how does the digest looks in your gel before the Southern transfert.

If the digestion is OK you should see an homogenous smear of DNA starting inthe high molecular weight down to the small size

pesji

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