The fragment cut by BamHI+XhoI is too short to show a difference between single and double digestion on gel, although you can use single digestions to verify that the enzymes can cut (but I am sure you already did that). 

To verify the double disgestion you could compare the uncut plasmid to the double-digested on gel; that should show a difference between the supercoiled uncut plasmid and your digested one, but that could in theory still be due to single cuts only, in case one of the enzymes does not work properly for some reasons.

Both XhoI and BamHI are pretty good enzyme though, so it is likely that the double digestion works efficiently even if you cannot visualize it. My suggestion would be to use a slight excess of enzymes, lets say 20-30 Units (each) for 3-5ug od DNA and incubate for 1-2 hours in a buffer containing BSA. Then column-purify to remove the small fragment and proceed with cloning. I usually do not dephosphorilate when using two enzymes, but you could do that if you prefer.

Finally, if you are really worried about partial digestions or religations, you could add a bit of a third enzyme with a restriction site between the previous two (lets say HindIII in this case) that would further digest the excised fragment and reduce the occurrence of these events.

Good luck

you should have on your gel the uncut plasmid, single digest for each enzyme, and the double digest for comparison.

The fragment cut by BamHI+XhoI is too short to show a difference between single and double digestion on gel, although you can use single digestions to verify that the enzymes can cut (but I am sure you already did that). 

To verify the double disgestion you could compare the uncut plasmid to the double-digested on gel; that should show a difference between the supercoiled uncut plasmid and your digested one, but that could in theory still be due to single cuts only, in case one of the enzymes does not work properly for some reasons.

Both XhoI and BamHI are pretty good enzyme though, so it is likely that the double digestion works efficiently even if you cannot visualize it. My suggestion would be to use a slight excess of enzymes, lets say 20-30 Units (each) for 3-5ug od DNA and incubate for 1-2 hours in a buffer containing BSA. Then column-purify to remove the small fragment and proceed with cloning. I usually do not dephosphorilate when using two enzymes, but you could do that if you prefer.

Finally, if you are really worried about partial digestions or religations, you could add a bit of a third enzyme with a restriction site between the previous two (lets say HindIII in this case) that would further digest the excised fragment and reduce the occurrence of these events.

Good luck

Hi,

Since both the enzyme produces cohesive ends, If it will be complete digestion by both the enzymes, after ligation and transform in bacteria, you will get no colonies. If it will be partial, just by any one of the enzyme, after ligation you will get colonies due to the self ligation.

Good luck.

@Amar,

Upon ligation, double-digested plasmids can form head-to-tail catenanes that get resolved by recombination upon transformation. If the competent cells are good enough and the amount of DNA is high enough, you will always get colonies in ligated, double-digested plasmids.

Hi Martin,

I am agree with you to some extent, but all the reactions are not 100% ideal. If someone is getting 2-4 colonies in double digest, I think It is OK. For more better results, the vector digested only with BamHI, and only XhoI, can be used as a control with the double digested plasmids in transformation reaction upon ligation. In double digested the chances of self ligation is very less as compared to single digested (BamHI or XbaI).  Then It can be confirmed whether double digestion reaction worked or not.

Thanks for comment

Amar's method works every time for me. I assume you are doing double digestion for the purpose of cloning. I always set up four ligations:

vector only, no ligase  - control for uncut vector

vector only, + ligase - control vector cut with only one enzyme

vector + fragment + ligase - your "good" ligation

fragment only + ligase - control for any problems with fragment

Good luck!

Hi Darius,

Thanks for supporting my answer.

Although letting this digestion go to completion is the best possible situation, it really isn't necessary.  Just dephosphorylate the pET21 backbone with alkaline phosphatase incorporated in the digestion.  Follow this up with gel purifying the cut-dephosphorylated vector, and ligate it with the complementary insert.  Perform a ligation control reaction with a ligation containing only pET21 cut/dephosphorylated/gel-purified vector.  This should yield no colonies when transformed in to competent E.coli.  Your ligation with both vector and insert should yield hundreds of colonies, optimally. 

You may, in practice, only observe tens of colonies however.  This all depends on your capabilities and the quality of resources you use.  Be sure to screen any colonies by PCR and/or restriction and sequencing. 

Best of luck.

I have done double digestion of vector Bam and Xho1 for 4 hrs in Cut smart buffer and it shows a single band in 0.6% agarose gel -i however did not do controls of single cut Bam and Xho 1 ?Could i gel purify and proceed for ligation??

purify your plasmid and then run 5ul of restricted and  non restrected plasmid and see if there is difference between them  .... then you can to proceed 

Sleman i already run an uncut control and the cut vector is clearly showing a difference  running exactly at the expected 5.4 kb

I could gel purify and run just supercoiled plasmid before proceeding for ligation

@Gautam,

First check whether you are getting any colony in double digested vector (only) upon transformation. If you are not getting any thing  and still you can see the double digested plasmid on the gel after purification, it means your double digestion reaction worked. For safe you can also check the single enzyme digested reaction in the same manner.

Good luck

Amar I do plan to use these controls you suggested.Last time around I got many colonies with the single cut vector but less in the double digest(technically should get nil) so i did not get recombinants

What controls do you normally do in yr cloning??Amar?

@Gautam,

First how many colonies you are getting in your double digested vector in compare to single digested vector . If you are getting 4-8 colonies it is Ok (some time), If you are getting more , It means your one of enzyme is not working good, change your enzyme or check by single digestion as per above discussion.

You can also enhance your timing of digestion.

I simply check the digested vector by transformation, If there are alot of colonies in double digested I will not use it.  

Good luck,

Amar

I got a huge number of colonies in the control double cut vector maybe around 100 this time around I am going to do what you suggested check by transformation

@Gautam,

It means your vector is not good . One of your enzyme is not working or has reduced activity. First check your enzymes.

Good luck.

Yes plan to do single digestions and optimise it before proceeding for ligation

commonly there is always some plasmid DNA can not be double-digested for some un-expected reason.

You can do gel purification to remove these un-digested plasmids.

If still can not sure if you get too much false positive clones, you can try this method:

http://www.sciencedirect.com/science/article/pii/S0003269714000888

Good luck for your work!

Quick and Dirty Directional Cloning

Cut vector with two enzymes and phosphatase in the same tube. I’d use 1ul of BamHI-HF, 1ul of XhoI and 1ul of rSAP in CutSmart buffer in 50ul for 1 hour at 37°C for 1ug vector. Purify the linearized vector with a spin column. This will usually get rid of the intervening sequence if the two enzymes cut in the polylinker.

If cloning a fragment from a PCR product, cut with the two enzymes and purify with a spin column. If the fragment is from another vector, you’ll want to gel purify and extract from a gel slice.

Quantitate the DNA and ligate 50ng of vector in a mix with an insert:vector molar ratio of 3:1.

Transform into the highest efficiency cells you can get your hands on and plate multiple dilutions. Transforming an equal amount of vector alone is a good control, but even with high background you can potentially identify successful insertion.

Screen 10 colonies by colony PCR using primers external to the cloning site looking for the correct size amplicon. Multimers are common with small inserts, but usually not an issue over 1 kb.

The methods for troubleshooting from this forum are good ones, but I wouldn’t troubleshoot until you run into trouble.

Ted I have a question why do I need to add phosphatase when it is directional cloning?

The second question is I have only insert primers to verify the colonies -did you mean that I should use a vector primer too?

Phosphatase will prevent any single cut vector from re-circularizing when you ligate, eliminating that as a source of background. Insert primers will work, but won't tell you if you have multimers.

Ted the strategy worked-thanks a ton -never believed it at first though when you suggested it