How to get rid of bands in PCR negative control
Posted 12 September 2013 - 08:38 AM
Posted 12 September 2013 - 11:09 AM
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.
Posted 13 September 2013 - 09:21 AM
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.
Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.
Posted 13 September 2013 - 09:09 PM
Posted 14 September 2013 - 06:01 AM
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.
Posted 15 September 2013 - 10:59 AM
Posted 15 September 2013 - 01:07 PM
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.
Posted 16 September 2013 - 07:52 AM
Posted 20 September 2013 - 04:07 PM
As phage434 mentioned, use something other than 70% ethanol - perhaps DNA away or 10 % bleach.
What is your downstream application with the amplicon? Do you run gels/visualize the PCR products in the same lab you set up the PCR? Are there others working with the same target?
There could be an issue with your work flow causing contamination of your wells with amplicon. This could also explain why you don't see contamination in every no template control (which should occur if it is a reagent issue).
As a simple test, perform a PCR reaction targeting a different gene in the same organism (preferably a target which is not regularly amplified!). If your negatives are clean, the contamination issue is likely due to amplicon resulting from your work flow.
Edited by biothreat, 20 September 2013 - 04:08 PM.