Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

How to get rid of bands in PCR negative control


  • Please log in to reply
10 replies to this topic

#1 Amena Baig

Amena Baig

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 12 September 2013 - 08:38 AM

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..???

#2 PhalanxBio

PhalanxBio

    member

  • Active Members
  • Pip
  • 18 posts
3
Neutral

Posted 12 September 2013 - 08:58 AM

Perhaps your pipets are contaminated?



#3 Amena Baig

Amena Baig

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 12 September 2013 - 09:06 AM

But I wipe my pipettes daily with 70%ethanol...

#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,467 posts
247
Excellent

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.



#5 Tabaluga

Tabaluga

    Making glass out of shards

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 392 posts
49
Excellent

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.

 


#6 Amena Baig

Amena Baig

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 13 September 2013 - 09:09 PM

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....

#7 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,467 posts
247
Excellent

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.



#8 Amena Baig

Amena Baig

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 15 September 2013 - 10:59 AM

I use new tubes from the box and Depc treated autoclave tips .

#9 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,467 posts
247
Excellent

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.



#10 Amena Baig

Amena Baig

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 16 September 2013 - 07:52 AM

I changed my buffers and primers dilution and surprisingly got band in one blank but not in other blank:\ I am confused now

#11 biothreat

biothreat

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

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.





Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.