How to get rid of bands in PCR negative control - (Sep/12/2013 )
I am getting band in blank exactly at the same position where my result bands are coming??I had successfully done my optimization and getting bands on required position without any band in blank but from last fews weeks I am facing this I have changed pcr master mix and water /??can anyone suggest what else I can do to get rid of this problem..???
Perhaps your pipets are contaminated?
But I wipe my pipettes daily with 70%ethanol...
That does nothing to eliminate DNA. More common is contamination of primers, buffers, water. You want to be using barrier tips if you are not already.
In your blank, is there just a band in the correct size or are there other, possibly weaker bands too ? The latter case would point towards aerosol contamination, while the former is indeed typical of a stock contamination, as phage434 said.
Yesterday I prepared 2blanks with the same components I.e with New primers dilutions,water,and pcr master mix and I am getting band in the 2nd blank and not in the first blank....???m still confused....
It could be your tips or your plates or tubes. Do you use the right from the box, or do you autoclave them? Autoclaves have lots of unexpected DNA in them.
I use new tubes from the box and Depc treated autoclave tips .
DEPC does nothing to eliminate DNA -- it's the opposite, inactivating nucleases. 70% ethanol does nothing to eliminate DNA. It sterilizes. Autoclaves do nothing to eliminate DNA. They sterilize, and probably add some DNA you weren't expecting. Dilute HCl, or bleach will have some effect. Use tips right out of the box. Don't do anything to them, unless you need them sterile. If you need them sterile and DNA free, order pre-sterilized tips.
I changed my buffers and primers dilution and surprisingly got band in one blank but not in other blank:\ I am confused now