Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > PCR - Polymerase Chain Reaction Forum
Register Search Today's Posts Mark Forums Read

PCR - Polymerase Chain Reaction Forum PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


pcr smear

pcr smear - PCR - Polymerase Chain Reaction Forum

pcr smear - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


Reply
 
LinkBack Thread Tools Display Modes
  #11  
Old 02-04-2009, 04:20 PM
Pipette Filler
Points: 10, Level: 1 Points: 10, Level: 1 Points: 10, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Post Re: pcr smear

Hello All
I am working on HIV pol gene. I am trying to amplify the gene since last few months. it was not working. Not the hot start is working. but it works for some samples for some time. when I used the same sample that previously worked, it worked once and then dint. For the other samples (new). there is big smear right from the well. I have tried the different sample size for the primary and secondary. It gives some samples worked at some concentration and some sampeles at another concentration.

Now I got the band in one sample which was bright but there was little bit smearing and others showed smearing right from the well.

The samples are already extracted and I cant redo the extraction as they are extracted in african country and then sent. so difficult to do the re extaction
Reply With Quote
  #12  
Old 02-05-2009, 02:43 PM
Pipette Filler
Points: 73, Level: 1 Points: 73, Level: 1 Points: 73, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: pcr smear

Hai,has anybody here dealt with asymmetric PCR before? What is the primer ratios that is suitable for asymmetric PCR?
Reply With Quote
  #13  
Old 02-05-2009, 03:59 PM
Aga's Avatar
Aga Aga is offline
Post-Doc
Points: 2,239, Level: 30 Points: 2,239, Level: 30 Points: 2,239, Level: 30
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2008
Posts: 270
Thanks: 25
Thanked 78 Times in 64 Posts
Default Re: pcr smear

Quote:
Originally Posted by cita View Post
Hai,has anybody here dealt with asymmetric PCR before? What is the primer ratios that is suitable for asymmetric PCR?
Actually it depends on the primer pair. I know I haven't helped you much
We used assymetric PCR several times in our lab and in each case the ratio was different. What actually are you trying to achieve? You can give me some general info so I may try to help, but still I think you'll have to test it empirically.
Reply With Quote
  #14  
Old 03-06-2009, 12:31 PM
Pipette Filler
Points: 59, Level: 1 Points: 59, Level: 1 Points: 59, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2009
Posts: 12
Thanks: 1
Thanked 0 Times in 0 Posts
Default Re: pcr smear

I am also facing this problem. I have tried to use the same DNA to do ITS, it worked well; but it always appear smear band on the AT rich region. I have tried so many times to exclude any possible mistakes, but failed. Who knows?
Reply With Quote
  #15  
Old 05-07-2010, 03:51 PM
Pipette Filler
Points: 3,383, Level: 38 Points: 3,383, Level: 38 Points: 3,383, Level: 38
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2008
Posts: 4
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: pcr smear

so... where is the solutions? just change the new DNA?
Reply With Quote
  #16  
Old 12-28-2010, 10:21 AM
Pipette Filler
Points: 1,340, Level: 21 Points: 1,340, Level: 21 Points: 1,340, Level: 21
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Oct 2009
Posts: 15
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: pcr smear

Hi
I have a problem in amplifying my gene . I did SOE and annealed the fragments using PR polymerase. but the annealed fragment was very less concentration. I used this as template and amplified using PR polymerase but getting a smear from well. what is the reason for this . I used ll polymerases and getting the same result.
Reply With Quote
  #17  
Old 01-11-2011, 02:36 PM
Pipette Filler
Points: 314, Level: 6 Points: 314, Level: 6 Points: 314, Level: 6
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2011
Location: manchester uk
Posts: 4
Thanks: 0
Thanked 2 Times in 1 Post
Default Re: pcr smear

I consulted the sage:
Smears the reason: Hydrolysis of the DNA making short DNA oligomers that act as Primers. DNA hydrolytic enzymes may be introduced by rogue bacteria or fungii. Work as clean as possible.
Why does one PCR will work perfectly and another inexplicably fail: Again bacterial DNA. In our case the PCR is to a phage insert so any contamination with bacteria that carry phage (all do) will bias the PCR towards smears.
Answer: "We" have managed to restore weak bands by re-extracting the tissue sample, do remove as much of the residual buffer so as to bias the PCR towards amplifying the gene of interest
Good luck.
Reply With Quote
  #18  
Old 03-30-2011, 09:59 PM
Pipette Filler
Points: 42, Level: 1 Points: 42, Level: 1 Points: 42, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2011
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: pcr smear

I am having a similar problem except I am just getting these rectangular blotches (grainy around the edges), and in one instance, I got my results but there were still these fuzzy squares directly below. Ladder is typically fine but it is doing that "smile" thing (leading edge seems to be migrating a little fast).
Reply With Quote
  #19  
Old 04-28-2011, 12:34 PM
Pipette Filler
Points: 314, Level: 6 Points: 314, Level: 6 Points: 314, Level: 6
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2011
Location: manchester uk
Posts: 4
Thanks: 0
Thanked 2 Times in 1 Post
Default Re: pcr smear

i posted on smears a while back.
after careful watching of what may go wrong I saw that the primers were placed in/on ice. The Ice is filthy with those black bacteria that reside in the water taps. Ask an electron microscopist. furthermore those black bacteria have been doing what comes naturally all over the lab especially tap handles. "Ok so how do they get into the PCR?" I hear you ask. Well, if at any time you handle screw top primers, or in fact any eppendorf tube, with wet gloves you will immediately syphon glove tip residue into the top of the tube. Leaving your primers in wet ice will result in the ice water creeping into the screw thread on the primers tube. enough said?!
Reply With Quote
The Following 2 Users Say Thank You to branthwaite For This Useful Post:
beginner (07-14-2011), danfive (06-21-2011)
  #20  
Old 06-20-2011, 10:56 PM
Pipette Filler
Points: 175, Level: 3 Points: 175, Level: 3 Points: 175, Level: 3
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: May 2011
Posts: 12
Thanks: 1
Thanked 0 Times in 0 Posts
Default Re: pcr smear

Hi, sorry to bother you all, but maybe you can lend me a hand for my problem

I am looking at the soil microbiota as a whole rather than isolating specific bacterial species. In my DGGE profile the intensities of each band is about the same at each lane
(seen at the same row), that's what make my head aches. I was hoping to see a different profile lane to lane, but it seems they were all look the same! But from one band to another in the same lane/column have different intensities. I used 338F-GC and 518R. Any ideas how can I avoid two bacterial species from stopping at the same point in my DGGE gel beside using different primers?
Thanks for your kind answer...
Reply With Quote
Reply

Tags
pcr , smear


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
AFLP smear Yuma248 Protocols and Methods Forum 0 08-13-2009 10:12 AM
Smear following Formamide/Formaldehyde denaturation of RNA cmcthorne RNA Techniques Forum 2 02-16-2009 09:21 AM
What smear bands mean? TRAN HO QUANG Protocols and Methods Forum 3 02-27-2008 10:03 PM
MSP product looks like some smear Amily Epigenetics Forum: DNA Methylation, Histone and Chromatin Study 2 10-16-2007 04:22 AM
PCR smear after CHIP dave2006 Epigenetics Forum: DNA Methylation, Histone and Chromatin Study 0 08-27-2007 02:53 PM


All times are GMT. The time now is 01:39 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.16941 seconds with 16 queries