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a big double digest problem

a big double digest problem - Molecular Cloning Forum

a big double digest problem - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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  #1  
Old 10-01-2010, 01:29 PM
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Default a big double digest problem



Hello people,

I have a continuos problem regarding a double digest of a vector of 10kB where the restriction sites are only one nucleotide apart. I've tried double digesting (BamHI-HF and XbaI) but it didn't work and when I tried to ligate and transform I had many false positives. I had colonies but they were empty so it ligated back which says that the vector was not digested by both but only one. so, now I have three different vector digestions:

1) first with BamHI and then with Xba
2) first with Xba then with BamHI
3) both at the same time

I ran them on the gel and purified them. after phosphatase treatment I will ligate them with a PCR product.

I made a PCR and added restriction sites to those ends for Bam and Xba. is there anything I should be careful with when I digest the PCR product? should I double digest or not? Shoul I purify the PCR product before digestion?

any comments, suggestions are very welcome.
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Old 10-19-2010, 09:51 PM
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Default Re: a big double digest problem

In your case I would first try double digesting (if the enzymes are compatible) your purified PCR product. If this doesn't work you still can make two single enzyme digests with your Insert.

If you have a spare plasmid from which you can cut out a significant big fragment with XbaI+BamHI, you can also make a digestion of it under the same conditions and use it as control for your digestion enzymes.
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Old 10-20-2010, 06:05 AM
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Default Re: a big double digest problem

Hola, Sorry if you have done it, I´mgoing to tell you how I done it;
Vector: prepare separatelly double amount of both digestions, incubate and take a part of them to check that the plasmid is linearized.If they are ok, mix and incubate again. Insert : purify the band in agarose gel and digest in the same way. After both digestion are finised, inactivate RE and mix to ligation in a molar relation of 1/5 vector/ insert. Transform and cross your fingers. Good luck
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Old 10-20-2010, 10:49 AM
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Default Re: a big double digest problem

thanks, it has been some time since I've posted this. right now it seems it is working. what i've done is I digested in 20ul with Bam or Xba and then I ve added the other enzyme and completed the reaction volume to 30ul. I gel purified afterwards,
made a shrimp alkaline phosphatase treatment, heat inactivated, ligated, dialysed the ligation rxn and then transformed the bacteria I've spread empty cells to one plate, concentrated and not-concentrated transformants to other plates. Interestingly, digestion with XbaI and then BamHI didn't work at all but digestion with BamHI and then XbaI worked )

cheers
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Old 03-24-2011, 07:23 AM
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Default Re: a big double digest problem

dear megalogaster,

today i did clone the PCR and tomorrow I will digest my PCR product and the vector pET25b before continue to the ligation process. Anyway, I've read your successful experience of digestion with these two enzymes BamHI-XbaI by separated digestion BamHI first then XbaI. My questions are:
1. how long u run digestion for firstly BamHI and which kind of buffer for it ? And for the next step, did you purified your digestion product (after BamHI) before continuing with XbaI? or you just directly add xbaI until the final volume 30 uL?
2. How about your digestion within the same time? Did it work? and which kind of buffer ?

Rgrds,
etin
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Old 03-29-2011, 02:18 PM
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Default Re: a big double digest problem

hi etin,

so, I run the digestion about 1,5-2 hours and I ran the one that has both enzymes around the same time. BamHI was HF enzyme from biolabs (NEB) so with buffer number 4. but then I've added BSA that was required for XbaI. I didn't purify although it would be the safer-cleaner way. the other one also worked.

i hope this helps,
best
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  #7  
Old 06-20-2011, 06:32 AM
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Default Re: a big double digest problem

hi
I have a doubt
what happens if the vector is partially digested .. will that vector works well or not ?
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Old 06-23-2011, 11:54 AM
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Default Re: a big double digest problem

hi
I think that is why you should make a control ligation. If your vector is not fully digested or digested but not CIP-treated then it tends to ligate back on itself. But I doubt that vector is not digested enough in an abundance of enzyme.
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