PCR water contamination - (Dec/21/2007 )
I have been trying to amplify TYMS primer, but each time, I get a band in my water (negative control), giving a fragment size the same as the patient DNAs and the positive control. The same water have been tested in other primers, and there is no band/contamination. We sequenced the PCR product from the TYMS/water control, BLAST the sequence, resulting in it aligning to TYMS gene. Any thoughts as to what is going on?
difinetly, it's contaminated
There are many sources of contamination u may use new PCR set and new set of pippettes.
But the most important thing is to purchase or prepare new PCR water.
I always prepare it by myself, divide it into aliquots and store at -20
PCR is quite sensitive, so one has to take care of all reagents used for PCR. Anything can be contaminated. Our lab had some problems with PCR contamination and we had to get new reagents every time there seemed to be contamination.
If its water, get new water. But make sure its water and not some other component of the PCR reagent.
yup, contamination has occured..... somewhere.....
The cure would be to throw everything away (dNTP, PCR buffer, PCR water, working primer stocks, tips and tip box, clean up the work surface (UV it if you a handheld lamp-remember to wear gloves and UV face shield) and get the pipette cleaned out.) and use new working stocks. It is often too much trouble to find which component has been contaminated. So throwing everything away and cleaning all surfaces and equipment is the fastest and least intellectually challenging method.
This is why it is important to make small aliqouts of everything that makes up your PCR kit. Contamination will occur given enough time, a single mistake or single aerosol droplet is all that is needed. Thus you want the ability to throw away everything you are currently using and pull up a fresh new vial of reagents without breaking the bank or more importantly have your boss come chasing after you for throwing away so much stuff.
Normally this is what i do:
Autoclave a bottle of water. aft that, alliquot in small eppendorf tube. everytime i do PCR, i'll just use the water fr the alliquot. After sometimes/if i suspect the water not clean, i'll just throw away the alliquot and take a new alliquot. If you scare your water will get contaminated leaving on bench, keep it in fridge (but i don't do it).
I don't really buy water for PCR. just autoclave distilled water and keep it sterile.