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.


Problem amplifying an insert from a vector

Problem amplifying an insert from a vector - PCR - Polymerase Chain Reaction Forum

Problem amplifying an insert from a vector - 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 09-24-2010, 11:19 PM
Pipette Filler
Points: 7, Level: 1 Points: 7, Level: 1 Points: 7, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2010
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Problem amplifying an insert from a vector



Hello

I am having problems amplifying a 1.9kb insert. I've done DNA sequencing on the vector to make sure that everything is there, aligned my primers to make sure that there wasn't some error with my primer design. I ran a positive control along with the sample. Positive control is good (1kb insert), nothing shows up for the sample of interest.

Here's the list of things going on in the reaction:

84ul water
10ul 10x vent buffer
1ul primer 1 (1uM final concentration)
1ul primer 2 (1uM final concentration)
2ul dNTPs (0.2uM final concentration)
1ng worth of template DNA (~1-2ul after appropriate dilutions)
1ul vent polymerase

for a total volume of ~100ul

Here's the conditions the PCR is run under:

1: 95C for 2min
2: 95C for 1min
3: 55C for 1min
4: 72C for 2min (I've actually tried doing the PCR reaction twice, doing it at 2min 30sec after finding out 2min didn't work)
5: cycle 2-4 29 more times
6: 72C for 10min

I think I've exhausted all the possibilities of what may have gone wrong with the PCR reaction and need your help guys.

Last edited by Dizon248; 09-25-2010 at 08:07 AM.
Reply With Quote
  #2  
Old 09-25-2010, 02:54 AM
jlf jlf is offline
Pipette Filler
Points: 380, Level: 7 Points: 380, Level: 7 Points: 380, Level: 7
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2010
Posts: 23
Thanks: 0
Thanked 6 Times in 6 Posts
Default Re: Problem amplifying an insert from a vector

How much primers are you using? 1uM works fine for small targets but I find that the larger the target, the lesser you have to use. Try going down to 0.2uM. Try reducing the number of cycles too. Excessive cycling lead to smearing and loss of product. Go for 20x first.
Reply With Quote
The Following User Says Thank You to jlf For This Useful Post:
admin (09-26-2010)
  #3  
Old 09-25-2010, 08:04 AM
Pipette Filler
Points: 7, Level: 1 Points: 7, Level: 1 Points: 7, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2010
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Problem amplifying an insert from a vector

I'll try reducing the concentration of primers. Although I don't understand why dropping primer concentration would help in this case.

I also don't understand the problem with my number of cycles as the issue with excessive numbers of cycle is loss of some product and smearing. I do not have ANY products. I'll try this also though... Thanks for the suggestions.

Again, all of these conditions used for the sample was used for a positive control which generated a band at expected size (1kb). Of course, there was smearing on my control as it is only 1kb and I ran it for 2min and 2min 30sec on second try.

Anymore advice would be great!
Reply With Quote
  #4  
Old 09-26-2010, 08:22 AM
jlf jlf is offline
Pipette Filler
Points: 380, Level: 7 Points: 380, Level: 7 Points: 380, Level: 7
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Sep 2010
Posts: 23
Thanks: 0
Thanked 6 Times in 6 Posts
Default Re: Problem amplifying an insert from a vector

I'm not certain how reducing primer concentration works but I can think of a few possibilities related to depletion of dNTP. Firstly, using too much primers will form more non-specific products which will expend dNTP. Secondly, if you look at how PCR works, the first few cycles will form many single stranded DNA that are longer than the target size. It is only when the reverse primer uses these new strands for amplification that the DNA of the correct size is formed. If you use more primer, more will anneal to the template and more long early DNA strands will form thus depleting your dNTP supply. By the time DNA of the correct size dominates, dNTP may already be low and cannot efficiently amplify your target product. Since larger targets require more dNTP, high primer concentration may be more detrimental for longer targets. Just my thoughts.

As for excessive cycles, maybe you HAD products but after more cycles, they have smeared out and become undetectable? Just maybe.

Also, I'm not sure if your denaturation and annealing times are too long but more recently, people are using shorter times for these steps. 15-30s is recommended by NEB for these steps. Saves time and reduces DNA damage. Try titrating MgSO4 concentration and adding 5% DMSO too.
Reply With Quote
The Following User Says Thank You to jlf For This Useful Post:
admin (09-26-2010)
Reply

Tags
amplifying , insert , problem , vector


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
Problem amplifying using colony PCR as template jlf PCR - Polymerase Chain Reaction Forum 3 09-24-2010 04:00 PM
problem in ligating 1.2kb insert in 9.4kb vector kumaarashwin Molecular Biology Techniques 1 09-19-2010 07:56 PM
vector ligation problem huazhongw Protocols and Methods Forum 0 02-26-2009 02:32 PM
PCR Amplification of Insert and Restriction Sites Problem admin PCR - Polymerase Chain Reaction Forum 2 01-25-2008 12:06 AM
DNA Cloning Protocol moleculardude Molecular Biology Articles and Protocols 2 08-05-2007 02:31 PM


All times are GMT. The time now is 04:15 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 0.13704 seconds with 16 queries