PRIMER DESIGNING PROTOCOL FOR

POLYMERASE CHAIN REACTION

 TO GET A GENE FOR WHICH A PRIMER HAS TO BE DESIGNED:

1) Go to web and search for UCSC GENOME BROWSER,

2) Get in to UCSC GENOME BROWSER and click GENOMES menu,
3) Type the name of the gene of interest at GENE textbox,
4) Give submit
5) Goto DNA menu
6) Now again repeat step 3
7) Click GET DNA button
8) Now click COLORS AND FONT OPTIONS button
9) Check all the boxes at the side of HUMAN MRNA’S option
10) And change the colour intensity no. in any of the color from 0 to higher value.
11) Now finally give submit.
12) Copy and paste the sequence in a word document.

 TO DESIGN A FORWARD PRIMER:

1) Select an exon of nominal amplicon size (some 400 to 500 bp),

2) Now select a forward primer at about 20-25 nucleotides long at about 16 nucleotides gap
from the exon,
3) Check for GC content (it should be above 40%)
4) Calculate the Tm value for the fp using the formula (2AT+4GC),
5) Now copy the sequence
6) Goto NCBI NUCLEOTIDE BLAST
7) Click the PASTE button
8) Click BLAST
9) Check for sequences it picks up (it should have at least 3 nucleotides difference from
sequence of interest)
10) Select it and browse for info about the gene,
11) If it is not closely related with the gene of interest, then, it can be used as forward primer.
 TO DESIGN A REVERSE PRIMER:

1) For the same exon, reverse primer should be selected in about 16 nucleotides gap from
the end.
2) Now one has to make reverse complement for the selected sequence(20-25 nucletides
long)
3) Now check the GC content for the reverse complement.
4) check whether the Tm value is ± 4o c with that of forward.
5) Now copy the reverse complement,
6) Goto NCBI NUCLEOTIDE BLAST
7) Click the PASTE button
8) Click BLAST
9) Check for sequences it picks up (it should have at least 3 nucleotides difference from
sequence of interest)
10) Select it and browse for info about the gene,
11) If it is not closely related with the gene of interest, then, it can be used as reverse primer.