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.


Error-Prone PCR for Mutation Library Generation

Error-Prone PCR for Mutation Library Generation - PCR - Polymerase Chain Reaction Forum

Error-Prone PCR for Mutation Library Generation - 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
  #1  
Old 03-02-2009, 05:47 AM
Graduate Student
Points: 2,354, Level: 31 Points: 2,354, Level: 31 Points: 2,354, Level: 31
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 114
Thanks: 19
Thanked 15 Times in 14 Posts
Default Error-Prone PCR for Mutation Library Generation



Dear all,

This is my first time over here in this forum and I would like to take the opportunity to get something sorted out.

I am trying to make a mutated library for selection. Basically, what I do is to do an error-prone PCR, followed by Dpn1 digestion and then an amplification to amplify the mutated PCR products left behind. This PCR products are cloned into a vector for transformation so sequencing can be carried out to characterize the library. The ligated products are also subjected to an amplification using biotinylated primers because I want a biotinylated mutated library.

Now, the problem is that though I do get occasional problems here and there for the PCR steps, they are often solved via different means. The step that is giving me a lot of problem is the PCR step using biotinylated primers. Whenever I do this particular PCR, I always get a smear in my gel results and I don't know why. I have tried diluting the template (which is the ligated products), changing the water, increasing the extension time, increasing the number of PCR cycles but none works. My partner in the same lab uses the same reagents as me for this particular PCR too and the only thing that differs between us is the ligated products, which is also our template and it works perfectly fine for her.

Do give me some advice as I have been at this for two months and time is running out for me. If this doesnt work soon, I will have to try another way of getting my mutated library but I would want this to work out because of all the work and effort I put into it. Thanks in advance!!!
Reply With Quote
  #2  
Old 03-09-2009, 01:55 AM
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: Error-Prone PCR for Mutation Library Generation

First, I want to ask you: do you have experience of good work on it? If you have, you can try to repeat that work in advace and see if it works well again.
You can ask your partner to do the same work(even the ligated product should be same ), and see if she can get the good results again.
Reply With Quote
  #3  
Old 03-09-2009, 05:29 AM
Graduate Student
Points: 2,354, Level: 31 Points: 2,354, Level: 31 Points: 2,354, Level: 31
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 114
Thanks: 19
Thanked 15 Times in 14 Posts
Default Re: Error-Prone PCR for Mutation Library Generation

Dear xinlong,

This project is new and I am the one who is starting on it. I have tried many ways to get the library but it doesn't work -- I still end up with a smear whenever I am at that particular step while my partner does not. It works perfectly fine for my partner and she uses the exact same reagent as me. Even my supervisor is really puzzled as to why it is not working when it should. I am unable to get her to use my ligated product and do it for me as she is busy with her own project.. sigh. I am trying out another method for these few days to see if it will work. Hopefully, I will get the library I want.
Reply With Quote
  #4  
Old 03-09-2009, 05:30 AM
Graduate Student
Points: 2,354, Level: 31 Points: 2,354, Level: 31 Points: 2,354, Level: 31
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 114
Thanks: 19
Thanked 15 Times in 14 Posts
Default Re: Error-Prone PCR for Mutation Library Generation

Dear xinlong,

This project is new and I am the one who is starting on it. I have tried many ways to get the library but it doesn't work -- I still end up with a smear whenever I am at that particular step while my partner does not. It works perfectly fine for my partner and she uses the exact same reagent as me. Even my supervisor is really puzzled as to why it is not working when it should. I am unable to get her to use my ligated product and do it for me as she is busy with her own project.. sigh. I am trying out another method for these few days to see if it will work. Hopefully, I will get the library I want.
Reply With Quote
  #5  
Old 03-09-2009, 01:54 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: Error-Prone PCR for Mutation Library Generation

I never suspect your operation on PCR. So, I wont give your some suggestion on operation, though some error operation can lead to smear bands. I think your ligated product is not good enough Because the only difference between you and your partner is the ligated product. On the other hand, you can do a negative control to see if there are smear bands in the negative control.
Good luck.
Reply With Quote
  #6  
Old 03-17-2009, 03:09 AM
Graduate Student
Points: 2,354, Level: 31 Points: 2,354, Level: 31 Points: 2,354, Level: 31
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 114
Thanks: 19
Thanked 15 Times in 14 Posts
Default Re: Error-Prone PCR for Mutation Library Generation

The negative control does not show a smear band, so I suppose the problem lies with the ligated product. I am trying to see what is wrong with the ligated product. But nevertheless, thanks a great deal for the discussion!
Reply With Quote
Reply

Tags
errorprone , generation , library , mutation , pcr


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
Natural selection is proven wrong gim Biology Forum 3 05-15-2009 07:43 PM
Assembly PCR, combinatoric library John Ladasky Protocols and Methods Forum 0 04-05-2007 07:56 PM
Optimal way to construct a YEAST 2-hybrid cDNA library? Apuzzlepiece@gmail.com Protocols and Methods Forum 0 08-10-2005 03:22 AM
pPC86 cDNA HeLa cell library Elena Schwartz Yeast Forum 0 04-26-2005 08:29 PM
RNAi library clone error reporting/bacterial contamination Julie Ahringer C Elegans Forum 0 05-19-2004 03:26 PM


All times are GMT. The time now is 11:41 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 0.14793 seconds with 16 queries