contaminations? - (Jan/08/2009 )
Good morning dear all.
I'm cloning a gene but when I sequence it I find an other one. this problem happened twice.
Unfortunately in our lab we haven't tips with filter.
all we use the same vector with inside the genes (with different length, 1.5 kb or 3 kb) which differnt people studies.
when we want to clone a new gene, we cut the vector (containing the past gene) always with the same restriction enzimes to re-use the vector and then we insert the new gene (modified and cutted with the same enzimes).
even if I'm sure to use my gene and the cutted vector in ligation, I found an other gene in sequencing..for example the gene of my lab neighbour.
it means that there are any contaminations?
How can i avoid this problem?
now I've repeated and I'm repeating the ligation but when I find positive colonies I'm suspicious to have the same problem descripted.
on my situatioin ,the only wway to verify the kind of gene inserted is to do PCR with specific primers, but with no results. problably because the vector plus insert is 6.5 kb?
Is possible to have so many contaminations and not to be lucky to find my expected gene?
Help me, thank U very much
well, since you are constantly reusing the same vector with the same restriction site, I would suggest that you make a base vector. Perhaps clone the lacZ gene into the vector using the restriction sites that you normally use. This would allow you to conduct a blue/white colour screen.
If you use this lacZ vector as a base plasmid for your cloning, it would help tell if your insert is in the vector or not just by doing a colour screen.
As for contamination, it shouldn't be a problem. My lab doesn't use filter tip either and we are fine. You insert will always be in abundance during the ligation. So upon recovery the probability of picking a stray contaminating plasmid is low. Unless the contamination problem is really horrible... ie people spilling plasmid preps all over your tips.
Still you should always grow up at least two isolates of the same plasmid and sequence both of them. So if one is wrong for some reason, you have a spare.
What concerns me is your inability to check your plasmid. Could a restriction digest not have picked up that the plasmid isn't the construct that you wanted? Or are you making mutants of the same proteins, so that is not possible? As for the PCR why shouldn't it work? The PCR should be made to amplify across the junction between insert and vector.
Maybe I'm also not a lucky guy, it happened in the past when I was trying to clone a plant gene but finally I got the E. coli sequence in my clones. It's so interesting because I used the specific primers but somehow, an E. coli strain was in my DNA sample and different fragments which had the same sizes as mine were amplified and I can not recognize them by both colony PCR and digestion (there fragments, unbelievable). PCR is very sensitive so sometime you can face this situation. Therefore, I think you should do some things: 1. confirm the vector (containing other gene, as you mentioned) from your peer, transform and select one colony and prepare the midiprep sample (so i assure the pure vector); 2: check your insert by PCR and digestion (complete digestion) to assure you have only one fragment); 3: also use unique digestion to check your clone before sequencing to save money and also your time. Good luck!!!