Bands in Negative control PCR - (Dec/05/2007 )
I think u should change ur PCR water
Some people use filter tips for pipetting PCR stuff to prevent contamination from pipettes.
We use filter tips to make our PCR mixes and aliquote these in the PCR-tubes, and we do so in a "pre-PCR" lab where we have to wear a labcoat specific for that lab and gloves. When all made and aliquoted into PCR-tubes, we can go the to "sample lab" (after taking of the lab coat, the gloves we don't change) where DNA and RNA extractions are performed and there we can add the template and in a next lab put them in the termal cycles. After cycling, we analyse on gel in a "post-PCR" lab. There is a strict prohibition on taking anything in the reverse direction between the labs without thorough decontamination with UV-light exposure (UV lamp is regularly checked) and each lab has a different set of pipettors. This might sound as over-doing this in order to prevent PCR-contamination but some of us are doing very sensitive nested PCR's on clinical samples from HIV-infected individuals so the last thing you want is contamination, so you do everything in order to prevent this. Everybody else has to follow this strict set of rules and this prevents a lot of contamination.
Most labs don't have space to do all of this so try to use filter tips (I know they are more expensive), use small aliquotes of water and primers (small aliquots are only used a few times, so this already reduces the risk of contamination by repeated use), have your samples open as short as possible and know that washing with ethanol won't help (think about it: ethanol precipitation is based on the fact that DNA is very poorly soluble to insoluble in 70% ethanol, how are you going wash it of with something it doesn't dissolve in?).
Most labs don't have space to do all of this so try to use filter tips (I know they are more expensive), use small aliquotes of water and primers (small aliquots are only used a few times, so this already reduces the risk of contamination by repeated use), have your samples open as short as possible and know that washing with ethanol won't help (think about it: ethanol precipitation is based on the fact that DNA is very poorly soluble to insoluble in 70% ethanol, how are you going wash it of with something it doesn't dissolve in?).
how to get rid of pipettes or tips contaminated with human genomic DNA? do you think UV light enough to get rid of it?
i asked my supervisor for separate set of pipettes, and he promised to provide me in one or two days. in the meanwhile i did two more negative PCRs. one from HPV6, HPv58 and beta actin primers for which i had bands in the previous PCR. But this time there were no bands for HPV6&58 but the bands for beta actin still there. i did not change anything but have different results. i did another negative PCR from the beta actin primers to check for third time using everything the same in one tube and in another tube just changed the water. again i got strong bands in both tubes. so the question is why were there bands for the first time in HPV6&58 negative control PCR and not for the second time using the same pipettes and every thing, but all the three times bands for negative control for beta actin? so what i am concluding is that the real culprits may be the pipettes but at the same time the negative results for HPV6 & 58 make me more confuse. whats your suggestions?
Thanx a lot
I can't say for certain why you are getting contamination sometimes and not at other times. My guess would probably be that there is dried DNA in your pippettor and sometimes the force of air knocks it into your tubes and sometimes it doesn't. I suppose that once you consistently get a good negative result using the same pippettor then you can say that the contamination for that particular gene is gone. But since you're getting your own set of pippettors anyway, i wouldn't worry about trying to clean up the other ones any more. Just make sure that when you do get your new pippettors that you keep them separate and don't load your PCR products with them.
have you cleaned the pipettes with bleach?
ethanol ill not get rid of the DNA contamination.
V
hi smu
i got new set of pipettes but the problem is still there. sometimes getting band and sometime not, using both old and new primer dilutions for beta actin as well as old and new set of pipettes, but for HPV 58 i have no more bands using each of the pipette sets. May be the liquid paraffin i am using in PCR has been contaminated with genomic DNA and also the tubes i am using for PCR are 0.5ml which sometimes dont fit very well in the wells and therefore sometimes i may get the bands and sometimes not, or may be all of my primers stock and dilutions contaminated with genomic DNA and the amount is very little so sometimes i get bands and sometimes not as someone already has commented in this thread, but if so why i am not getting bands for HPV58 anymore except for the first time? what you will suuggest? thanx everyone for suggestions and advice.
Hi vetticus3
what sort of bleach do you mean? is there some special one? i want to know that even now i have new pipettes but may be it work out.
thanx a lot
i got new set of pipettes but the problem is still there. sometimes getting band and sometime not, using both old and new primer dilutions for beta actin as well as old and new set of pipettes, but for HPV 58 i have no more bands using each of the pipette sets. May be the liquid paraffin i am using in PCR has been contaminated with genomic DNA and also the tubes i am using for PCR are 0.5ml which sometimes dont fit very well in the wells and therefore sometimes i may get the bands and sometimes not, or may be all of my primers stock and dilutions contaminated with genomic DNA and the amount is very little so sometimes i get bands and sometimes not as someone already has commented in this thread, but if so why i am not getting bands for HPV58 anymore except for the first time? what you will suuggest? thanx everyone for suggestions and advice.
Hi vetticus3
what sort of bleach do you mean? is there some special one? i want to know that even now i have new pipettes but may be it work out.
thanx a lot
Hmm...I really thought that getting new pippettes would clear things up a lot. Maybe you need to order new primers and start with all fresh materials. Also what someone else suggested about setting up different labs for different steps in the process will help. If you don't have the space to do this then maybe designate a particular area in your lab just for doing the set-up steps. Also maybe getting some DNA away and filter tips will help too.
As a side note, I know that you're probably getting very frustrated at this point, and rather than burn yourself out I suggest that you take a couple days off from doing these experiments. It will help you clear your mind a bit and give you a much needed break.
Sorry I can't be of more help.
smu
Thanx a lot smu for nice suggestions especially the side note. I am really very frustrated and my supervisor also asked me the same as you suggested, to have a rest for few days. In the meanwhile i also confirmed that the oil i add to the PCR reaction was not the cause of contamination, as that lab has a hot lead PCR and dont need oil to be added. the results were the same as before, as the preparation for PCR i performed in my own lab. Anyhow Thanx once again and have good time.
007