I have been trying to digest a vector carrying an mCherry tag using SgrBI (as an alternative to SacII), but I am failing in my attempts. The reaction that I set was as follows: 6ul SgrbI buffer (the enzyme and buffers are inhouse products, not from a commercial source), 0.6ul BSA (100x , and from NEB), a 3ul of the vector , 4ul of the enzyme and made to 60ul with water. The reaction was carried out for 2 hours at 37. Any suggestions?
Yes Markus, I know the concentration of my vector .. it is a 1ug/ul
you could also check by sequencing if the restriction site is really there. Did the digest work with SacII?
well i have the map of the vector which says the site is there as a unique one , and it is only because I do not have a SacII exactly, that I am opting for SgrbI
Just to make sure the enzyme is actually working, maybe you should try digesting another vector containing SacII sites... then you can figure out if the problem is the enzyme or your plasmid preparation
Is your vector being prepared from a bacterial source (e.g. miniprep of E. coli bearing your plasmid), or some other source? And what are you using to determine whether or not the reaction worked? Running out the digest product on an agarose gel, or are you putting this directly into another reaction?
Jamie, yes my vector is from a bacterial source ( a medi prep) and I have been running the digest on a gel to determine the reaction.
Guillermo I did find this information last evening, but I was wondering if a prolonged reaction time like 16 hours at 37 might help?! I have gone for it and right now I am running a gel to check it. I will keep you guys posted.
How old is your enzyme? Enzymes stored for long time and in and out from freezer for many times sometimes wont work. Ensure that your enzyme is fresh and concerned buffer. In case if you are using NEB enzymes check the specifications before application. Most of the RE should around 37C and incubate O/N. Check your plasmid prep as well. Ensure that there are no impurities. Better to use TE buffer to obtain or dissolve the plasmid pellet rather than other buffer. Otherwise use distilled water.
Check all these and try.
Good luck
Aditya
Aditya thank you for the suggestions! But I have already made sure of all that you have mentioned , save for the fact that I do not use an NEB enzyme, it is an inhouse product., still I did try using fresher enzymes and buffers from a central stock and neighbouring labs too with no luck. The conditions were set just as they were recommended. The plasmid is fine and Im sure because the second restriction enzyme (BamHI) worked just fine.
Now what I am actually considering to do is try with the actual SacII from NEB, since so far it is SgrBI ( an isoschizomer for SacII) from the inhouse facility that Iv been using which did not yield any results.
guys thank you all for your valuable thoughts and suggestions! my digestion finally worked when I tried with a new aliquot of SacII . the enzyme stocks that I had been using last week apparently had the problem. I guess they must have gone old or lost activity for some reason.. anyways now that I have my digest I am proceeding with my cloning.. Thanks a lot again!
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