Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Molecular Biology Techniques
Register Search Today's Posts Mark Forums Read

Molecular Biology Techniques Molecular Biology Forum. Includes forums for common molecular biology techniques.


PCR amplification problem

PCR amplification problem - Molecular Biology Techniques

PCR amplification problem - Molecular Biology Forum. Includes forums for common molecular biology techniques.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 03-26-2008, 05:03 AM
Pipette Filler
Points: 822, Level: 16 Points: 822, Level: 16 Points: 822, Level: 16
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2008
Posts: 14
Thanks: 0
Thanked 0 Times in 0 Posts
Default PCR amplification problem



I have to amplify a gene of size >=3.5 kb, from an unknown genome.

my limitations:

using the usual taq
i'm using the cDNA thats 6 months old (from RACE kit)


how do i get the amplification??!!?
Reply With Quote
  #2  
Old 03-26-2008, 03:04 PM
danfive's Avatar
Nobel Laureate
Points: 8,612, Level: 64 Points: 8,612, Level: 64 Points: 8,612, Level: 64
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2007
Location: Houston TX
Posts: 969
Thanks: 12
Thanked 184 Times in 154 Posts
Default Re: PCR amplification problem

I would go straight to a TOPO vector.
I did a couple projects using them and have no complaints.
Also the kit was only a couple hundred bucks, not expensive at all (big time-saver).
Reply With Quote
  #3  
Old 03-27-2008, 06:16 AM
Pipette Filler
Points: 822, Level: 16 Points: 822, Level: 16 Points: 822, Level: 16
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2008
Posts: 14
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: PCR amplification problem

dear danfive, thankyou for your reply, but the problem is that i haven't yet reached the stage of cloning, i'm yet to know why i cannot get the pcr product right... all i get to see is the dye or smear in the gel.

so any suggestions??
Reply With Quote
  #4  
Old 03-31-2008, 07:34 AM
Pipette Filler
Points: 1,804, Level: 26 Points: 1,804, Level: 26 Points: 1,804, Level: 26
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2008
Posts: 29
Thanks: 0
Thanked 2 Times in 2 Posts
Default Re: PCR amplification problem

I think its written in literature that normal taq amplifies fragments upto 2kb properly. However I had a gene that was also 3.5kb long which i could amplify easily with usual taq. You could try ussing Pfu polymerase (amplifies bigger fragments, but leaves blut ends..), also the other problem could be the primers.. please check the primers properly..(bioinformatically.....there are many hitches there..)..also other Thing could be degarded DNA but I think you would have ruled that possibility out already...
Let me know when you get the amplifiaction ... and the reasons behind it.. I am really curious about this one...(Could help me also..)
Cheers
Priyanka
Reply With Quote
  #5  
Old 04-01-2008, 05:07 PM
Pipette Filler
Points: 822, Level: 16 Points: 822, Level: 16 Points: 822, Level: 16
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2008
Posts: 14
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: PCR amplification problem

thanks priyanka... i'm chking the cpndintion of the cDNA... its an interesing problem for me too... i serched and there are a few othe taqs that can be used for longer fragments ... genaxxon taq, kapa2 taq.. etc.. i'll let you know he details afetr the amplification.. ... and blunt ends is what i want
Reply With Quote
  #6  
Old 04-02-2008, 03:38 PM
Pipette Filler
Points: 1,804, Level: 26 Points: 1,804, Level: 26 Points: 1,804, Level: 26
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2008
Posts: 29
Thanks: 0
Thanked 2 Times in 2 Posts
Default Re: PCR amplification problem

I hope that you dont forget to get back with the details...
I thought may be I could Ask you for a favor... iam isolating RNA these days.. At first the RNA was completely degraded... But today the gel shows 4 RNA bands and smearing between the bands..( i think its not the best quality RNA) I would like to know some precautions/ tips that I could follow for the same.. I tried IRIS the first time.. today however i used Trizol...
Reply With Quote
  #7  
Old 04-09-2008, 08:38 AM
Pipette Filler
Points: 822, Level: 16 Points: 822, Level: 16 Points: 822, Level: 16
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2008
Posts: 14
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: PCR amplification problem

hey, sorry for the late reply... you are on the right track i should say. but whats the source of your RNA? is it plant RNA or animal...?? if you can send over a gel picture, i'll be able to analyze it in a better way. good luck.
Reply With Quote
  #8  
Old 04-09-2008, 02:14 PM
oBWhat's Avatar
Graduate Student
Points: 3,826, Level: 41 Points: 3,826, Level: 41 Points: 3,826, Level: 41
Activity: 50% Activity: 50% Activity: 50%
 
Join Date: May 2006
Posts: 124
Thanks: 0
Thanked 7 Times in 7 Posts
Default Re: PCR amplification problem

Hello Priyanka,
the smearing either sounds like bad RNA preparation or degradation in some steps.

Have you tried any RNA isolation kits ie QIagen or wizard?

Reply With Quote
  #9  
Old 04-10-2008, 05:39 AM
Pipette Filler
Points: 1,804, Level: 26 Points: 1,804, Level: 26 Points: 1,804, Level: 26
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2008
Posts: 29
Thanks: 0
Thanked 2 Times in 2 Posts
Default PCR amplification problem

I am working with plants... and Intrestingly trizol is giving little better results as compared to IRIS..( I have no idea why).. and kits are not something that my teacher recommends.. so they are out of question... the problem is still that Iam getting diffused bands of almost all sizes but along with a lot of smear.... And what about your gene (3.5kb)
Reply With Quote
  #10  
Old 04-10-2008, 02:37 PM
Pipette Filler
Points: 1,804, Level: 26 Points: 1,804, Level: 26 Points: 1,804, Level: 26
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2008
Posts: 29
Thanks: 0
Thanked 2 Times in 2 Posts
Default Re: PCR amplification problem

I just thought i`ll let you guys know I got beautiful RNA profile with no background or anything, Perfect amount of smear (mRNA) and needless to say I am really relaxed now..
The problem I could identify was very bad Agarose quality...and bad ethanol...Thankx guys for all the help..All the Best!!
Cheers
Priyanka
Reply With Quote
Reply

Tags
amplification , pcr , problem


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
2D PAGE problem antheab Peptide Forum 3 12-26-2010 04:28 PM
Weird amplification curve for Taqman probes fen Real-Time PCR and Quantitative PCR Forum 0 01-25-2010 09:35 AM
weird problem in transfecting 293 cells in 24-well plate Cell Biology and Cell Culture 1 04-17-2007 03:54 AM
N-body problem Torstein Raanes Physics Forum 1 12-01-2003 10:31 AM
Integration Problem / Finding Contour lines Problem blah12@mail.com Physics Forum 2 09-07-2003 11:28 PM


All times are GMT. The time now is 03:28 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 0.17088 seconds with 17 queries