Water and cloning? - Major cloning disfunction... (Dec/15/2006 )
Hello,
We're having some serious cloning problems in the lab at the moment.
We tried to test various parameters, starting from the obvious ones : restriction enzymes, ligation enzymes, transformation process, and nothing seems to be bad...
So basically, we're down to a "water problem", knowing that everything that we used is derived from milliQ water produced in the lab, but there is a suspicion that maybe the machine is not very well taking care of (things that are supposed to be changed might not have been...).
So basically my first question is : could that make sense???
The thing is that to test the ligase, we tried to digest a plasmid, purify it on gel and re-ligate it, and that work beautifully...
So my second question is : does anyone have any suggestions about other things that we could test???
Thank you so much for your help!
if the millipore system is not cleaned or parts replaced , then one could have contamination or impurities in the water which could b the culprit.
After a few more tests, it appears that it's not due to a water problem...
So we're left with the idea that something wrong happens when we prep the DNAs, knowing that we digest them and then purify them with the qiagen kits...
Does anyone have anything to suggest, please???
contamination from the quigen kit.
Are you gel purifying your DNA bands after the digest? (Does the RE digest work?)
Contamination from either the Q buffer or the PE buffer (EtOH) is inhibitory to ligation.
Are you gel purifying your DNA bands after the digest? (Does the RE digest work?)
Contamination from either the Q buffer or the PE buffer (EtOH) is inhibitory to ligation.
Yes I'm sure that the RE digest works, and yes I'm gel purifying my DNA bands.
And I do an additional 5 minutes spin after the PE buffer in order to get rid of all the ethanol before the elution, if that's what you're thinking of...
contamination from the quigen kit.
Are you gel purifying your DNA bands after the digest? (Does the RE digest work?)
Contamination from either the Q buffer or the PE buffer (EtOH) is inhibitory to ligation.
Yes I'm sure that the RE digest works, and yes I'm gel purifying my DNA bands.
And I do an additional 5 minutes spin after the PE buffer in order to get rid of all the ethanol before the elution, if that's what you're thinking of...
Yes.
And before that spin step, do you leave the PE buffer to soak on the column for 2mins?
EDIT: Oh and one more thing, what restriction enzyme are you using? In my experience ends cut by SalI ligated very poorly for some reason. I have read on NEB website that some rarer restriction enzyme also have similar problems... they produce unligatable ends.
*Note- NEB says nothing about SalI being a bad enzyme.
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Yes.
And before that spin step, do you leave the PE buffer to soak on the column for 2mins?
EDIT: Oh and one more thing, what restriction enzyme are you using? In my experience ends cut by SalI ligated very poorly for some reason. I have read on NEB website that some rarer restriction enzyme also have similar problems... they produce unligatable ends.
*Note- NEB says nothing about SalI being a bad enzyme.
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No, I don't do the soaking step, I never have and my cloning used to work, but I will definitely try that!!
The enzymes that I used are very common enzymes (HindIII and BamHI) that should work well, and someone else in the lab is trying an EcoRI cloning which is also not working.
I'm not trying to do anything fancy, just a simple cloning of different PCR fragments (so it can't come from the insert, because their different and all the cloning are not working) in a plasmid that I used a lot before (with the same enzymes by the way!)...
Thank you so much for trying to help!
If you are gel extracting, do you expose your DNA to short wave UV? Has someone changed the setting on your transilluminator from long wave to short wave, and have you failed to notice?
We have a specially old transilluminator to cut the DNA, and it's impossible to change anything on it :-). But I will check if it's a long or short wave setting... And I'm very careful to expose the gel for the shortest time possible...
do u use the same kit for everbody use?? i mean do u use the same container of kit ??
if everybody (more than 20 ) of lab use the same container , there is a chance of contamination because everyone is not equally careful