Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Designing primers for the ORFs

ORF cloning primer design

  • Please log in to reply
9 replies to this topic

#1 sujatharaghu

sujatharaghu

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 19 January 2012 - 01:20 AM

Hi,
I'm sujatha.I need some help in designing primers for a particular gene. I have to clone this particular gene GDXL lipase from Aeromonas, now when i give the sequence of this gene in NCBI ORF finder,I'm getting a result such that only in the non template strand the entire gene is covered( my gene has abt 921 bp, only the -1 reading frame covers the entire gene frfrom 1-920 and also the starting codon is TAG and the 920 th codon is ATG. Now can i reverse this sequence and design primers specific for this ORf? Also am planning to use topo vectors, can some one pl guide me as to how to go abt designing primers and wht aare the factors that i should be considering for designing primers with restriction sites which is to be cloned in topo vectors?

#2 biocrazy

biocrazy

    member

  • Active Members
  • Pip
  • 21 posts
1
Neutral

Posted 19 January 2012 - 02:47 AM

For Topo cloning whatever the gene (or with adaptor), you just need to add CACC to it.

#3 sujatharaghu

sujatharaghu

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 19 January 2012 - 07:00 AM

thnk you.....but can u tell me more abt orf interpretation?

#4 biocrazy

biocrazy

    member

  • Active Members
  • Pip
  • 21 posts
1
Neutral

Posted 19 January 2012 - 05:42 PM

Don't worry about ORF bcos when you do LR reaction with destination vector, only the sequence what you had in the PCR product will be transfered to destination vector.

#5 sujatharaghu

sujatharaghu

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 23 January 2012 - 03:49 AM

Thank you biocrazy,
so, you mean to say after I retrieve the gene sequence in FASTA format from NCBI, I can manually design primers for the forward and reverse sequence or take help from any primer designing software ? I have another question, how do i ensure that my primers are apt for my gene of interest before actually doing a PCR? Is there way to cross check using primer blast? Correct me if I'm wrong. Thanks again......

#6 biocrazy

biocrazy

    member

  • Active Members
  • Pip
  • 21 posts
1
Neutral

Posted 23 January 2012 - 06:02 AM

That you can check using any program. You are right you can use primer blast and any other online tools.

Here is a link, you can find many useful thinks.

http://www.bioinformatics.org/sms2/

#7 sujatharaghu

sujatharaghu

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 25 January 2012 - 01:41 AM

Thank you bio crazy for the tool

#8 biocrazy

biocrazy

    member

  • Active Members
  • Pip
  • 21 posts
1
Neutral

Posted 25 January 2012 - 03:26 AM

You are welcome Sujatha. If you don't mind, may I know where you are working?

#9 sujatharaghu

sujatharaghu

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 26 January 2012 - 07:59 AM

hi,
this is a public form so wudnt want to say all that.... whats your name and what do you work on?

#10 biocrazy

biocrazy

    member

  • Active Members
  • Pip
  • 21 posts
1
Neutral

Posted 26 January 2012 - 05:27 PM

If you wish mail to satha.selvam@gmail.com





Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.