hi
Can any one help me to find out what is the reason that i get bands in my negative control (no DNA template) while doing PCR. Sometimes i get no band while on the same day another time i get very bright bands in the negative control. i am using paraffin embedded tissue DNA extracted by phenol choloroform method. i want to check the integrity of extracted DNA and for that purpose i am using beta globin, beta actin and GAPDH primers. i never got good results for beta globin althogh the proimers i am using have been used by many others and i have selected them from those articles, but for beta ctin and GAPDH i sometime get very good results with no bands in negative control but for another time when i do PCR from the same samples i get bands in the negative control as well. i tried all the possible ways to get rid of it for the last two months but in vain. i tried to autoclave everythings like tips, pcr tubes, wash the pipets with alocohol, autoclaved double distilled water freshly but on the same day the first PCR has no bands in negative control but the second one has bands in negative control. I am really very tired of it and cant find any possible reason.please help me.
Good Bye
Thanx in Advance

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I'll say check your primers. Make new aliquots or even get new stocks. I had similar problem recently, with b-actin and GAPDH too, and was all in the primers. The reason sometimes you get it and sometimes you dont is probably due to the fact that your primers are contaminated with a small amount of DNA that sometimes will amplify but sometimes will fail to do so.

I work in a pretty "dirty" lab, and my only source of contamination was the primers. of course is worth trying fresh reagents, and each of the old ones with everything else new, by my bet is on the primers.

Good luck!

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check you pcr water, pipettes or aerosol contamination,.....any of these cud be the culprit ......first of all start by using a new set of pipettes for aliquoting

hope this helps

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This is a very common problem with PCR and occurs because PCR is very sensitive and it takes only a small bit of DNA to contaminate your solutions, pipettors, bench, etc. Most typically I find that the contamination is in the pippetors. In other words, you run your PCR products out one day, a small bit gets stuck in the pippetor (don't ask me how) and acts as a template for the next time you do PCR. To get around this, I typically use two different sets of pippetors - one that I consider to be "clean" - it has never seen my template, and the other "dirty" which has. Always set your reactions up with clean pippetors, then use your dirty one to load and run your gel. Always include a negative control (like you obviously have), which doesn't have any template in it as this will tell you if you have contamination. Barrier tips can help too, although I've also gotten contamination with these as well.

Hope this helps.

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the fist 2 things you have to do is:
1. Fresh water.
2. Fresh primer dilution.

although... i once had contamination from the PCR buffer. So, there is a simple check list to go through, BUT the main culprits are the water or the primers.

V

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Hi
thanks all once again. I tried to prepare new stocks and new dilutions with new PCR water and PCR mix (beads), using new room for preparing for PCR but the same pipetts as used by the other students as its not possible to have separate ones but washed with alcohol. Again i got very strong bands in the negative control tube. so then i tried to check all the primers with the same water and PCR beads as used in previous PCR but no DNA template in any of the tubes, in other words i did a negative PCR for 10 pairs of primers for HPV6,11,16,18,31,33,45,58,beta actin and GAPDH. so again i got very strong bands in some of the tubes namely HPV6,58 and beta actin, but in one tube where i got band in the previous PCR for HPV58, no band was found this time. interestingly the bands in each case were of the size of the real PCR product. so what is the possible reason behind this. what should i do further?.
Thanx in advance
Bye

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Going back to your original statement, you say that you want to check the integrity of your DNA, meaning that you want to know that you have good quality DNA. I'm not sure that I would use PCR to check for the quality of your DNA as PCR can be very sensitve and can work even if your DNA quality is poor. What are you using the DNA for? Is it ultimately for a PCR experiment? Can you use some other method to check it?

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Thanx somu2
i am extracing DNA from paraffin embedded cancer tissues and will use it for HPV typing. for this purpose i have to find out whether the negative samples are really negative or there is no DNA at all. so first i have to make sure that there is DNA by using beta catin or GAPDH.
secondly i can check the DNA by gel electrophoresis but its not enough to get band on the gel. also i tried to check it by spectrophotometer but the amount is just negligible and in some cases the same readings as for empty cuvett (water in which the DNA was dissolved). so the only way is the internal control PCR to check the DNA integrity.
But now i am facing another problem as i mentioned in the second post.
thanx

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I see, much clearer now. I've done a lot of quantitative RT-PCR in which a negative control has to be purely negative. Whenever I get contamination, it has always been solved by changing the pippettors like I suggested before. Assuming that you've changed every reagent in your PCR reaction and use clean tips and tubes, then really this is the only possible source of contamination that I can think of. I'm not certain if merely washing them with ethanol will get rid of contaminating DNA. You may have to think about getting some DNA away (available from several vendors). It may be a convention in your lab that everyone shares pipettors, but for your purposes, I think that you have to be a little bit aggressive and claim a set as your own. Otherwise if others are using the same pippettors for the same PCR reactions, they will keep getting contaminated and you will continue to have the same problem. Use them ONLY for setting up the reactions (use a common set for loading). Another alternative is to go to a lab that doesn't do PCR on these genes and borrow their pippettors.

Finally, if your spec readings are inconclusive, you can try to increase the amount that you use to measure in the spectrophotometer. When the readings are close to zero, it can be difficult to say with any certainty that what you have is real. By increasing the concentration ten fold, you can be more certain (assuming of course that you have enough DNA to do so).

BTW- its really good that you're including the negative controls. I know many people (even profs!) who don't do these controls, and I wonder how they can ever be certain of what is real.

Good luck to you.

smu

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Thanx smu
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

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