PCR products sizes and DNA ladder - (Nov/30/2013 )

I am working on Vitamin D receptor gene polymorphism using Fok I and Taq I and I am using, I am having trouble in determining PCR products sizes of (TT, Tt, tt, FF, Ff, ff) and the PCR marker/DNA ladder.

Taq I: 5’-CCT TCT TCT CTA TCC CCG TG-3’ (Forward) and 5’-GCA GGT CGG CTA GCT TCT-3’ (Reverse)

Fok I: 5’-ACT CTG GCT CTG ACC GTG-3’ (Forward) and 5’-TCA TAG CAT TGA AGT GAA AGC-3’

 I have looked through too many articles, what should I do?

Thank you in advance

Best regards  

The easiest way would be to get the full sequence of the gene from the NCBI site, and use primer BLAST or something similar to find the primer binding sites - then you should be able to work out the predicted length of the amplicons.

bob1 on Sat Nov 30 21:03:41 2013 said:

The easiest way would be to get the full sequence of the gene from the NCBI site, and use primer BLAST or something similar to find the primer binding sites - then you should be able to work out the predicted length of the amplicons.

thank and sorry for the late response

could you be more detailed

Go to the NCBI website (www.ncbi.nlm.nih.gov) - search for your gene, choose a refseq for your species (these are the reference sequences against which all other submitted sequences are compared and should be full length).  Get the sequence or the accession number (NM_XXXXXXXXX, where XXXXXXX will be a number) for the sequence.

Go to primer blast (www.ncbi.nlm.nih.gov/tools/primer-blast/).  Enter the details required. 

bob1 on Thu Dec 5 20:11:16 2013 said:

Go to the NCBI website (www.ncbi.nlm.nih.gov) - search for your gene, choose a refseq for your species (these are the reference sequences against which all other submitted sequences are compared and should be full length).  Get the sequence or the accession number (NM_XXXXXXXXX, where XXXXXXX will be a number) for the sequence.

 

Go to primer blast (www.ncbi.nlm.nih.gov/tools/primer-blast/).  Enter the details required. 

http://www.ncbi.nlm.nih.gov/snp/?term=vitamin+d+receptor+gene

this link shows the results that I got, how can I determine which one for my primers?

thank you in advance

YOu need to choose the nucleotide option from the front page.  You can limit to species as well.  It's your project - you should know which snp it is...

bob1 on Sat Dec 7 20:43:42 2013 said:

YOu need to choose the nucleotide option from the front page.  You can limit to species as well.  It's your project - you should know which snp it is...

my specie is homo sapiens and I know the rs for both primers :

  taqI :rs 731236

fokI:rs 2228570

and this is the link after I chose nucleotide

http://www.ncbi.nlm.nih.gov/nuccore/?term=homo+sapiens+vitamin+d+receptor+gene

Ok, now choose to limit by Refseq (right hand side, under "Filter your results")  Choose the variant that applies to you...