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

cDNA Forum cDNA Forum. Discuss complementary DNA synthesis, cDNA libraries, and cDNA cloning in this forum.


Troubleshooting 5' and 3' RACE using Clontech SMARTer RACE cDNA Amplification Kit

Troubleshooting 5' and 3' RACE using Clontech SMARTer RACE cDNA Amplification Kit - cDNA Forum

Troubleshooting 5' and 3' RACE using Clontech SMARTer RACE cDNA Amplification Kit - cDNA Forum. Discuss complementary DNA synthesis, cDNA libraries, and cDNA cloning in this forum.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 12-01-2010, 05:47 AM
Pipette Filler
Points: 63, Level: 1 Points: 63, Level: 1 Points: 63, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Dec 2010
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Troubleshooting 5' and 3' RACE using Clontech SMARTer RACE cDNA Amplification Kit



I'm having problem of getting any band for my 5'- and 3'-RACE using Clontech SMARTer RACE cDNA Amplification Kit. I had try to re-isolate my RNA, synthesis new cDNA and re-designed new gene specific primers, but still cannot get any positive result.
Any suggestion for my problem?
Reply With Quote
  #2  
Old 09-23-2011, 11:59 PM
Pipette Filler
Points: 110, Level: 2 Points: 110, Level: 2 Points: 110, Level: 2
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2011
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Troubleshooting 5' and 3' RACE using Clontech SMARTer RACE cDNA Amplification Kit

I am having the same problem did u figure out the problem
Reply With Quote
  #3  
Old 11-08-2011, 02:21 PM
Pipette Filler
Points: 82, Level: 1 Points: 82, Level: 1 Points: 82, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2011
Location: London
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Troubleshooting 5' and 3' RACE using Clontech SMARTer RACE cDNA Amplification Kit

Hi,

I'm just beginning to do my gene cloning sequence using the RACE technique. I was wondering what protocol you followed to check the quality and yield of the RNA?

Also does anyone have any idea what the maximum mRNA transcript size that RACE is able to amplify? My full length gene transcript is 4.6bp. Would this be a problem.

I'll be happy to share any tips and suggestions once I get started.
Reply With Quote
  #4  
Old 11-08-2011, 02:32 PM
Pipette Filler
Points: 82, Level: 1 Points: 82, Level: 1 Points: 82, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2011
Location: London
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Troubleshooting 5' and 3' RACE using Clontech SMARTer RACE cDNA Amplification Kit

Hi,

I will be using the First Choice RACE kit to obtain the full length sequence of my gene. I've got one question. Does anyone what the maximum length of the mRNA transcript that can be cloned using this kit? My mRNA is about 4600bp and I'm wondering whether it will be able to catch the extreme 5' and 3'UTR sequences.
Reply With Quote
  #5  
Old 11-16-2011, 07:47 PM
Graduate Student
Points: 1,231, Level: 20 Points: 1,231, Level: 20 Points: 1,231, Level: 20
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jun 2011
Posts: 113
Thanks: 0
Thanked 16 Times in 13 Posts
Default Re: Troubleshooting 5' and 3' RACE using Clontech SMARTer RACE cDNA Amplification Kit

I think there isn't an absolute limit on how large a transcript you can clone, this probably depends entirely on how well your RT step works.

In my hands RACE is quite messy (I never get a clear band, the product is always a bit smeary) so I really only use it to map the very ends of transcripts (alternative promoters for instance). For determining the sequence of your mRNA of interest it may just be easier to order several sets of primers across the RNA, do RT-PCR and sequence the products (or just look for splice variants by product size on a gel).

Unless you're working on a species for which there is no transcriptome data, I would say spend some time looking at UCSC genome browser or Ensembl and then design primers to amplify the full length transcript by conventional RT PCR.
Reply With Quote
Reply

Tags
amplification , cdna , clontech , kit , race , smarter , troubleshooting


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
Rapid Amplification of cDNA Ends question? hianghao PCR - Polymerase Chain Reaction Forum 3 02-21-2010 04:13 PM
5' RACE troubleshooting Yarri Protocols and Methods Forum 0 01-26-2009 11:28 AM
cDNA Synthesis and Amplification of POLY(A) mRNA by RT-PCR Protocol domba Real-Time PCR and Quantitative PCR Forum 3 09-09-2008 02:48 PM


All times are GMT. The time now is 08:41 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 0.13624 seconds with 16 queries