Argggg! Contamination - its eating me alive!!! (Sep/07/2006 )
Hi there to all,
Im one of the victim's of the usualy universal problem . NEGATIVE CONTAMINATION!!!! I have no ide how to get rid of the negative band. I have pratically done everything in the book to overcome this problem but nothing seem to prevailling. Im using the universal primers for 16S rDNA. the primers are 519f n1492r. The base size is 973bp. Pls help. I cant solve the problem. I have changed the water, the reagent, but nothing seems to prevail. My personal thought is that its the primer because the 519f binds universally and the 1492r binds only to bacteria. Mayb the primer is the culprit. Is there anyone whom using this primer which face the same problem.
P.s. The first lane is the negative, wherelse the other lanes are the plasmids which will be sent in for sequencing after RFLP. The negative lanes show the same band size as the other plasmid.
Pls help. Will appericiate any help.
Thank loads!!!
If you've changed EVERYTHING except your primers it suggests you've got some template DNA contamination in one or both of your primer solutions.
Try making making up new solutions from primer stock.
are you setting up your PCR's in another lab space? preferably out of the room? do you use dedicated pipettors and tips? do you change your lab coat? I know the theory of 'DNA floating around in the air' is considered whack by some people, but it really can make a difference; especially if you frequently amplify the same gene(s) in the same space
There are reported cases of bacterial genomic DNA contamination in some enzyme preps (they are made in bacteria, after all). How many cycles are you running. Bacteria are everywhere, and you can amplify just one of them, given enough cycles. I'd suggest that you might find success by running a bit more of your template, and doing the PCR for only 20 cycles. An easy experiment is to set up a negative control and take samples at 15, 20, 25, 30, 35 cycles to see when it amplifies. Easier, if you have access, is to do it in a quantitative PCR machine with sybr-green PCR mix.
Of course, it could just be a contaminated reagent. You need to change *everything*. Water, tips, tubes, primers, enzymes, buffers, dNTPs. I'd highly recommend barrier tips. Clean your pipettor barrels. Don't autoclave tubes and tips -- use them straight from the sealed box.
I set up my PCR in the same working space. Our lab doesnt have separate pipetor for PCR. Anyway i have 2 other lab mates whom do PCR also but with different primers. They seem to not to have any problems in their PCR.
Try making making up new solutions from primer stock.
i alwis make new solutions from my stock when ever i do my PCR. But still the same.
If you don't have separate room for PCR master mix preparation, at least you should have a hood specially for PCR. Further, when I re-constitute or prepare my primer stocks, I use filter pipette tips. Of course, I perform everything in PCR room. In addition, make sure you have your individual sterile and autoclaved water for PCR. No sharing of reagents, if this is possible.
Hi,
I prepare my pCR in the special hood and also my water is separate. i dont use filter tips and thinking of purchasing some. Do you think it will make a different using the filter tips
depends on your level of care during pipetting.
I don't use filter tips or even a special set of PCR pipettes or even a special place for PCR. Heck, I prepare maxipreps on the same bench with the same set of pipettes that I use for PCR... actually it is amazing that I am not hideously swamp by contamination, coming to think of it.
The key thing is to assume that no matter the level of care and deligence, everything, from your reagents, to your tips, to your lab coat, to your gloves will become contaminated.
So sub aliquote all the reagents (100ul) (in somebody else's lab, perferably one who works on something else). Change water for every PCR reaction, pipette slowly, use a new tip box and keep the gloves on a top shelf. And consider making your primer solution a very dangerous operation. Great care must be take, treat it like you would a radioisotope. Don't breath on it. And dump the lab coat. Your daily clothes would probably be cleaner then the lab coat... when was the last time the old coat was washed?
But I guess when all said and done. Filter tips would be a good thing. So many people use them.
I had a situation with bacterial DNA contamination in an Taq enyzme prep. I was using qPCR for neo resistance gene (same seq. as kan resistance gene). I did every control and changed every reagent possible. This went away only when I changed enzymes companies. You may consider trying Taq from a different company or lot number.