disappearing vector after digestion - (Jun/19/2006 )
After double/single digestion of my vector, it just disappears and i´m left with a smear at the bottom. I have tried without pcr purifing it, so i know that´s not the problem. The insert is digested just fine with the same enzymes in the same buffer...This is puzzling everybody who has seen it...I have run the undigested plasmid and it looks just fine. I have cut plenty of plasmid (1ug) so there should be lots to se. I even cut just 200ug to see if that would help, but still the same results... Anyone with a possible solution?
The REs are PstI and EcoRI and Im using buffer H from TaKaRa (as suggested from the manufactors)
edit: the vector that im using is the pMal-c2x straight from the kit, so there should be nothing wrong there
may b one of the components of the vector digestion mixture is contaminated. did u use the same solutions to digest the insert n vector?
Try to incubate ur DNA with the buffer and no enzymes and then run it on the gel. Also try some other vector as control.
everything was completely identical. I should mention that the smear is just bearly visible, it is not bright at all...Seems strange that something could completely digest the vector, but not touch the insert..
May b try to digest the vector again. Hopefully it works.
I know that some DNA preps might have some exonuclease carry over during purification (from some strains of E.coli) , but this is not a problem if u had purified the DNA initially thru column.
good luck !!!
I know that some DNA preps might have some exonuclease carry over during purification (from some strains of E.coli) , but this is not a problem if u had purified the DNA initially thru column.
good luck !!!
Already tried it twice, that´s why i´m so frustrated...
what exact conditions have you tried? could you be getting star activity from the Eco RI? are you using more enzyme or cutting longer than with the insert digestion?
So if you digest insert and vector seperately and the insert looks ok afterwards and the vector is degraded then you might have DNAses in your vector prep. They are active during the restriction digestion (I don´t know: maybe for 2 hours at 37 °C) and cut you vector. The uncut plasmid that you run on the gel has not been incubated at 37 °C and so it is not degraded much.
Just incubate several ul of undigested vector at 37 °C for 1-2 h and see if the DNA gets degraded or not. If it is, do a new vector prep.
Chalet, if that is indeed the problem, could you not save a bunch of time and effort by a simple P:C:IAA extraction?
You could incubate your plasmid just in water to see if the problem is your prep or something also.
I would do F: C extraction and elute in a different (TE or water).
I would do F: C extraction and elute in a different (TE or water).
I just have incubated this sample and another vetor in the cutting buffer for 1 hour at 37 and they showed up. Then i cut again, purified and nothing...
A260 says there is 19.5ng/ul in the sample, so it should show on the gel.
There cannot be a nucleases in the vector, because i used it straight out of the kit from NEB.
Have tried cutting with one or two enzymes as well without any difference, so that´s not it either.
The restriction enzymes are EcoRI and SalI