The plasmid may contain another site you are unware.

Are you sure the plasmid is what it said it is? Did anyone successfully construct with this plasmid？I think you should digest it BamHI and BsaI respectively to see if there are two sites of one of them.

I checked but there is no site in between gene of interest and only one site is present for BamH1 and Bsa1.

Next to all of the above, I would also check if your enzyme is not showing star activity.

I thought BamH1 is an enzyme that has that. Nowdays the newer enzymes should  not have these problems, and there are HF (High Fidelity ) enzymes that eleiminat this star activity.

Star activity is the fact that your enzyme can cut similir sequences unther sub optimal conditions, thus giving a different gel outcome.

As Ziguo Zhang suggested, "The plasmid may contain another site you are unaware.”

1. There might be an error in the DNA sequence data which omits a site for either BamHI or BsaI.

2. Or, in particular, the plasmid may contain a second site for BsaI that wasn’t detected due to an error within the computer program used to find sites for BsaI.

Because the recognition site for BsaI is non-palindromic, both strands of the plasmid sequence must be analyzed. Within computer programs for analysis of recognition sites of restriction enzymes, you can usually depend upon the accuracy of the analysis. But, rarely, inaccurate results occur when programmers input data for restriction enzymes having non-palindromic recognition sites, such as BsaI.

For BamHI (and other restriction enzymes which recognize palindromic sites) the programmer needs only to input one recognition site: GGATTCC. Regardless of which strand is analyzed, the analysis will reveal all recognition sites for BamHI.

But, when searching for sites for BsaI (or any other restriction enzyme which recognizes non-palindromic sites) the programmer must input the recognition site in both the forward orientation and backward orientation. He must input both sequences: GGTCTC and CCAGAG. You can perform this search manually to confirm the number of recognition sites for BsaI within your plasmid. [NEBcutter performs this search accurately.]

3. It is possible that the unexpected band resulted from Star Activity (i.e., cutting by either BamhHI or BsaI) at sites resembling their cognate recognition sites. Both BamHI and BsaI are prone to Star Activity. And BamHI is likely to exhibit Star Activity when using a low-ionic-strength buffer recommended for BsaI. Simultaneous digestion is very likely to result in loss of specificity by BamHI.

If you perform a double-digestion using BamHI and BsaI then it should be done sequentially, not simultaneously. First digest with BsaI (using the recommended conditions) then add NaCl to 150mM, and digest with BamHI.

-paul walsh

Have you tested for Star activity?  You could accidentally be altering the reaction composition if you add a large volume of DNA containing unintended salts, contaminants or alter the pH.  Similarly, excess enzyme will add too much glycerol, which can also lead to star activity.   https://www.neb.com/.../what-is-star-activity-and-how-can-it-be-avoided

Hey Akshay, 

The things mentioned above are likely the reason you aren't seeing the expected "two bands." On another note, do you expect to see two bands as you stated or three bands? 

Best, 

David

It is possible that the unexpected band results from unexpected restriction site on the backbone of the plasmid. You may have an insert on the plasmid like a transposon. In order to check this, you should digest the plasmid with 3 or 4 restriction enzymes together and see were you get your unexpected band, or unexpected size of expected band.

All good input, particularly the non-palindromic issue in programs and star activity. NEB's BamHI-HF does pretty well at avoiding star activity. There's also the possibility that mutations could have occurred elsewhere in the backbone of your vector. This is not an infrequent problem. You can digest vector and construct singly in each enzyme to be sure you're only coming up with predicted band to be sure this is not your issue. You could also sequence, but the digestion is probably cheaper and faster. If you get predicted bands when doing single digests, then you'll know the issue is not mutation in vector or incorrect detection in program (which you can also check manually as Paul Walsh suggested above) and could likely just be star activity.

Hi, i've ever faced similar problem with you when i did restriction mapping project. Have you ever tried to do single digest for each of restriction enzyme that you used in your plasmid sample? Then, it should be followed by double digest enzyme. So, you have 3 samples to be run in electrophoresis for the visualization. after that, i hope that you can see where is the problem :) 

I agree with David. Why do you suppose to see 2 bands? According my experience, you should get minimum 3 bands. Find out why.....? Do some reference studies. 

My plasmid is circular so i supposed to get 2 bands not 3 @ David @Jai.

Akshay,

You will get two bands at the very least, as circular plasmid DNA undergoes supercoiling, but what about the linear DNA present after digestion? The appearance of only two bands is unlikely.  

Which competent E. coli strain have you used? BsaI is blocked by dcm and CpG methylation. You could try to use a dam–/dcm– competent E. coli.

Akshay,

There are three possible answers to your question;

1. The third lower DNA band may be undigested DNA. In order to be sure this isn't an undigested DNA band take as control the undigested vector during gel electrophoresis and compare the DNA bands provided by the undigested vector with this band.

2. If the third lower band looks like a smear it might be RNA contamination. In order to verify wether it is an RNA contamination, I want to know wether you isolated the DNA plasmid yourself?  During DNA isolation did you use a resuspension buffer containing  RNAse or any other RNAse containing solution?

3. The third lower band might be as a result of star activity if you digested your DNA samples with these restriction enzymes for a long time. Lets say more than 3 hrs and above.

Could very well be contaminating bacteriophage. Happens more often than one might think. Happened to me, and gave a strong band a bit small than a kb. The band is often brighter than larger bands, indicating a non-stoichiometric ratio to your plasmid-generated bands. You can probably ignore it for most purposes, if cloning. I don't know how to get rid of it from plasmid preps, as re-transforming infects the new cell also. In the future, use a bacteriophage-resistant e. coli strain if possible.