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


primer concentrations in PCR

primer concentrations in PCR - Basic Lab Protocols and Techniques

primer concentrations in PCR -


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 10-10-2007, 12:24 PM
Pipette Filler
Points: 986, Level: 17 Points: 986, Level: 17 Points: 986, Level: 17
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Oct 2007
Posts: 3
Thanks: 0
Thanked 0 Times in 0 Posts
Default primer concentrations in PCR



I'm currently working on cloning the gene for CFTR, a chloride transporter found in gills, intestine and in a few other organs, but I am having trouble getting anything on a electrophoresis gel after running a PCR, and I suspect it has something to do with my PCR.

I have three sets of degenerative primers and two sets of conserved primers that I have to choose from, but I am currently using them at a low concentration (10 microM working solution). I have also tried running a dilution series on my cDNA, but with no luck

Have any of you out there attempted running a PCR with a higher concentration of primers than I am currently using? And if you have, did you shorten the annealing time? And did you leave the annealing temperature unaltered, or did you change it? And what concentration cDNA did you use?

Hope someone wants to share previous experiences with someone who did not taker a molecular education, but who is working with it now!!
Reply With Quote
  #2  
Old 10-10-2007, 03:40 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: primer concentrations in PCR

I responded to your other post concerning primer concentrations, so I'll ignore that here.
I worked with PCR from gills, as well. They are not very difficult but you should consider that the gills may contain a lot of blood and the iron in the hemolymph/globin is a PCR inhibitor---solution to problem: cut small sections of gill (i.e. 0.1g) and soak in pure water for 20 minutes. Take your tissue and just about any DNA extraction procedure will work and give you good quality DNA from there. To be extra secure you can use a Clontech Chromaspin Column-100 to purify your DNA a step further.

PCR is a great tool. Keep posting questions and I'm sure you'll get all your primers giving you PCR products real soon.
Reply With Quote
  #3  
Old 11-15-2007, 06:30 AM
Pipette Filler
Points: 884, Level: 16 Points: 884, Level: 16 Points: 884, Level: 16
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2007
Posts: 10
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: primer concentrations in PCR

10uM is good. changing the concentrations is not going to have much of an effect. The thing that will help the most is trying to optimize the annealing temperature. Hopefully you will have a PCR machine where you can run multiple samples with different annealing temperatures to quickly test this.

Otherwise, a primer redesign is in order.
Reply With Quote
Reply

Tags
concentrations , pcr , primer


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
Second Guessing myself on primer concentrations nebulus PCR - Polymerase Chain Reaction Forum 1 05-01-2010 07:49 PM
Bad primer design ? How can I know ? surt Bioinformatics 4 10-07-2009 07:24 AM
I need immediate help!! How to PCR primer designing? :help: veniesh PCR - Polymerase Chain Reaction Forum 2 08-20-2009 04:00 PM
How primer concentration can me measured? Kamalian, Laleh Protocols and Methods Forum 0 12-16-2008 01:17 PM
please help me in primer dimer saly Real-Time PCR and Quantitative PCR Forum 0 09-06-2008 02:18 PM


All times are GMT. The time now is 09:23 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 0.12683 seconds with 16 queries