9 replies to this topic

[sujatharaghu]

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?

[biocrazy]

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

[sujatharaghu]

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

[biocrazy]

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.

[sujatharaghu]

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......

[biocrazy]

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/

[sujatharaghu]

Thank you bio crazy for the tool

[biocrazy]

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

[sujatharaghu]

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

[biocrazy]

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