I don't think it's star activity either - I've used excess amounts of EcoRI or left it overnight at 37 and it has never caused me any problems. Besides, if it was an issue of non-specificity you wouldn't be getting two bands but rather all three, and maybe some more (there can be other similar sites). Better sequence your DNA, and/or change enzymes. Still, if you think you can get enough of the 1300bp band to try and purify it from a gel, try reducing the incubation time (try 30 mins for example) and the enzyme concentration, and make sure the condtions (temperature and buffer) are optimal for it. Even if you manage to do your cloning (?) like this, be sure to sequence the final product, as this is a suspicious behaviour and all sorts of things could go wrong (even to the point of having the wrong DNA, which could cost you months of work at some later point). Better be safe than sorry!

Hi Mahesh..

The problem you are pointing out may not be exactly 'star activity' as mentioned by you...BamHI and EcoRI are very commonly used enzymes for a quite long time..the assays of these enzymes are well optimized and they show star activity only if your are 'unnecessarily using ' more than recommended amount of enzyme or incubation time....Check if your assay is as per the recommendem conditions.

The second opinion is the observation may not be result of star activity ...there may be actually a EcoRI site presnet in the site...this can be a possibility ...if in the sequence which you are using (if it is from database) some error may be present right from the begining...or...the sample/strain/species you are suing may be different thanfor which the sequence is present in the database (so some variation may be present including a new site for EcoRI)...check these issues along with the incubation time and amount of enzymes used

Try reducing the enzyme concentration or increase ionic strength of buffer

@ Ajay soni, Thank you sir for your valuable suggestion. I confirmed by using Webcutter 2.0 and Nebcutter that gene is not cutting in between but the EcoR1 enzyme is recognising the sequence "GAATC" in between the gene. Actually the restriction site of EcoR1 is "GAATTC".

I don't think it's star activity either - I've used excess amounts of EcoRI or left it overnight at 37 and it has never caused me any problems. Besides, if it was an issue of non-specificity you wouldn't be getting two bands but rather all three, and maybe some more (there can be other similar sites). Better sequence your DNA, and/or change enzymes. Still, if you think you can get enough of the 1300bp band to try and purify it from a gel, try reducing the incubation time (try 30 mins for example) and the enzyme concentration, and make sure the condtions (temperature and buffer) are optimal for it. Even if you manage to do your cloning (?) like this, be sure to sequence the final product, as this is a suspicious behaviour and all sorts of things could go wrong (even to the point of having the wrong DNA, which could cost you months of work at some later point). Better be safe than sorry!

Hi Mahesh

I am just curious to know...from where you have got this gene sequence....have you done it your self....or from some company...in both these case some times careful analysis of sequence electropherogram has to be done ..some times the sequencing machine see a 'slightly broad' peak (of two TT, GG, AA or CC) as 'single base' but when you see the profile carefully and also see the sequence conservation the base position should have two bases...that one has to do by manual editing....WEB CUTTER will analyze only the sequence you are feeding in it...it will not tell what thesequence is exactly......

if the sequence is from database.......don't be very confident ....mistakes can be there too by the people who originally submitted the sequence.......if they were not carefull during the submission of the sequence...it is seen often......

in any case there is a significant probability that the sequence you are seeing as 'GAATC' in the gene and visualising it as a site for star activity of EcoRI may actually be 'GAATTC"......if you have a sequence profile with you check it carefully or send it to me...

I think you need single digestion. First cut your DNA with BamH1 for 2-4h and apply PCR purification for removing the BamH1 buffer(of course you will lose some DNA). Then digest it again with EcoR1 for short period(not over night) and check in gel. Good luck.

Hi, you can use NEB restriction enzymes master mixes (okay, there are only a few available...): http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/landing_REMix.asp#.T3mdGdnVYzk

@ Ajay Soni Sir, I reduced the incubation time from 60 min to 10 min. And I found same result i.e 3 band(1very light band of 1.3kb, 2nd band of 900bp and 3rd band of 400bp.) I have taken gene sequence from database NCBI.

Hi Mahesh, I would suggest you then to equence the plasmid insert by yourself with any standard sequencing primers (M13, T3 resp. T7 etc.) whichever fit for your plasmid backbone and then you will probably see some mutations which are not present in the original sequence from the NCBI database. If you find mutations you might think of mutate them back anyway (in vitro mutagenesis kit from Invitrogen) since they might influence your gene of interest.

It sounds like the sequence you have from NCBI is wrong then, and you need to change your strategy (meaning substitute EcoRI with another enzyme). If this is a PCR product and EcoRI is at the 3'end of the gene do a Klenow, purify, cut with BamHI and try to clone (we are still assuming you are cloning the gene) as BamHI-blunt. If it's a bigger fragment, or even better, an already cloned fragment, find another suitable enzyme from your sequence (it's highly unlikely there will be two mistakes in it). Even better, if it's an already cloned fragment, sequence it. You may also be able to sequence a PCR product using the primer from your pcr, but this depends on your sequencing method. And finally you MAY want to try a partial digestion (like you did before, 5 or 10 minutes only, then purify the 1300 band from a gel) to get your fragment BUT DO NOT try this if it's a PCR product you are cutting, as you won't be able to know if the 1300 bp is indeed cut with both enzymes at its' ends. You can only do it if you are isolating from another vector, so the uncut DNA will be a totally different size. It would be helpful at this point to know exactly what you are trying to do.

I think your problem is not with star activity but there is an internal EcoRI site in your insert. My suggestion is first you confirm the sequence by gene sequencing and also select different restriction enzyme will be better.

@ Dr. Vathsala, Sir u r right...Its better to change the restiction site in primer and restriction enzyme.