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PCR products sizes and DNA ladder


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#1 sayk

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Posted 30 November 2013 - 03:24 AM

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  

 



#2 bob1

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Posted 30 November 2013 - 01:03 PM

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.



#3 sayk

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Posted 04 December 2013 - 04:28 AM

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



#4 bob1

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Posted 05 December 2013 - 12:11 PM

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. 



#5 sayk

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Posted 07 December 2013 - 03:21 AM

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....d receptor gene

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

thank you in advance



#6 bob1

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Posted 07 December 2013 - 12:43 PM

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...



#7 sayk

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Posted 07 December 2013 - 01:01 PM

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....d receptor gene



#8 bob1

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Posted 12 December 2013 - 12:11 PM

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






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