A quick & basic guide for primer design for amplification of Genomic DNA:
Good primer length
Annealing temperature
GC content
~24 bp.
~600C.
usually less than 50%.
A good internet site for designing primers:
http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi
For primers to be a good primer pair, they need to have similar qualities (Tm, GC etc)
Increased length increases their specificity.
They should not anneal to each other and should have minimal secondary structure.
These things can be checked manually or by feeding the primers sequence into a program
such as 'GeneRunner' – can be downloaded from http://www.generunner.com/ or by
letting a program like primer3 design your primers for you.
Primer3
This is the program's main window
Step1 – paste the sequence you want to amplify in PCR into the window.
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Step2 – add '<' and '>' and the beginning and end of the region you want to be amplified
(sequence that should later be sequenced or just for limiting the sequence for a minimal
length of an amplified fragment).
For example:
If the sequence is
TGGTAGAGACATGCCCCTACCGCCCTGATTGGCTGAAGCTGCGTGCCTGGTGAGGTGACGTGGCTTGCTGTG
AGTGGTTGGGGGCTGAGAGTATATAAGAGTGAGAGATCCGGTGATCGAGGGAGAAAAAGATGAAGCGAGAGA
GATGAAGATTGAAGCTTGCTGACTAAACTGCTGTTAGAACTGGTGGTCGTGTCGTTCTTGCTGGTTGAGAGC
AGACACGACAGTTGCCTACGAGAATGCGATAGTAAGACTGCATTGCTGAAGACAGCACACATCTTGGTCACA
GAATATAAAGAAATAAAACTGAAACTGGCCTGGGAGCGTCCTCCCCATTAGCTAGCCATCATGACGCATCAA
TACAACTGACTACTCAGGCATAATATAGTGCAAGAGTGGCCAAGTTTGCCGGGCGTGGTGGCACACGCCTTT
AATCCGAGCACTCGGGAGGCAGAGGAAGGCGAATTTCTGAGTTCAAGGCCAGCCTGGTCTACAAAGTGAGTT
CCAGGACAGCCAGGGCTACACAGAGAAACCCTGTCTCCAAAAACCAAAAAAAAAAAAAAAAATAGTGGCCAA
GTTTGTTCCAGAGGTAAGCAACCACTTTCTGATTGGATTTGAAGCCAATCCTATGAAGGGATTCCTCTCTGA
TACTGTAAATCTGGTCAAATCCCATGGTTGGAAAGGCTATAGGCCCTAGTTGGGAAGCTACGCTGTTCTTTT
GGTAAATAGACATCATGTACCCATCAAATTGAAATTAATTAAGTGGGGCTTTTATAGGCCTATGTCTCTCCT
TGCTGCTGGATTAATGCTCCTAAGATAACAGGGGGGAAAAAAACTCTCTGCAGGATTGTTGAACTTCGGAAC
CTCCCTGTCCCAGACAGATTCCTCCTTGGAAGTGGATCTTTTGCTGCAACTCTGTGTCTGATCGATTCCCTT
TTAAGGGATAAAAGGACTTGGAATCCCTCTAGGGGGAATGCTCTGAACTGCAGTATCAACTGATAACAA
And I'm interested in the sequence in Red. Then '<' and '>' should be added before and
after this sequence – for example –
TGGTAGAGACATGCCCCTACCGCCCTGATTGGCTGAAGCTGCGTGCCTGGTGAGGTGACGTGGCTTGCTGTG
AGTGGTTGGGGGCTGAGAGTATATAAGAGTGAGAGATCCGGTGATCGAGGGAGAAAAAGATGAAGCGAGAGA
GATGAAGATTGAAGCTTGCTGACTAAACTGCTGTTAGAACTGGTGGTCGTGTCGTTCTTGCTGGTTGAGAGC
AGACACGACAGTTGCCTACGAGAATGCGATAGTAAGACTGCATTGCTGAAGACAGCACACATCTTGGTCACA
GAATATAAAGAAATAAAACTGAAACTGGCCTGGGAGCGTCCTCCCCATTAGCTAGCCATCATGACGCATCAA
TACAAC<TGACTACTCAGGCATAATATAGTGCAAGAGTGGCCAAGTTTGCCGGGCGTGGTGGCACACGCCTT
TAATCCGAGCACTCGGGAGGCAGAGGAAGGCGAATTTCTGAGTTCAAGGCCAGCCTGGTCTACAAAGTGAGT
TCCAGGACAGCCAGGGCTACACAGAGAAACCCTGTCTCCAAAAACCAAAAAAAAAAAAAAAAATAGTGGCCA
AGTTTGTTCCAGAGGTAAGC>AACCACTTTCTGATTGGATTTGAAGCCAATCCTATGAAGGGATTCCTCTCT
GATACTGTAAATCTGGTCAAATCCCATGGTTGGAAAGGCTATAGGCCCTAGTTGGGAAGCTACGCTGTTCTT
TTGGTAAATAGACATCATGTACCCATCAAATTGAAATTAATTAAGTGGGGCTTTTATAGGCCTATGTCTCTC
CTTGCTGCTGGATTAATGCTCCTAAGATAACAGGGGGGAAAAAAACTCTCTGCAGGATTGTTGAACTTCGGA
ACCTCCCTGTCCCAGACAGATTCCTCCTTGGAAGTGGATCTTTTGCTGCAACTCTGTGTCTGATCGATTCCC
TTTTAAGGGATAAAAGGACTTGGAATCCCTCTAGGGGGAATGCTCTGAACTGCAGTATCAACTGATAACAA
Now this sequence can be pasted to the programs window. It's enough to paste it with 2-4
lines of sequence before and after the sequence of interest.
Step 3 – setting parameters
The program has default parameters that are better to change. This is the parameter
window:
2
Product size – choose the size you need. If the sequence you have entered between the
brackets is 300 bp in length, you set product size to minimum 320, maximum 450.
Primer size - Min 23
Opt 24
Max 26
Primer Tm -
Min 58
Opt 60
Max 60
Primer GC -
Min 20
Max 50
Step 4 –
Picking primers – click on the 'pick primers' button.
The following output will show
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the first primer pair is usually the best one.
The program calculates also the product size.
By scrolling down additional primer pairs will show.


If no primer pairs show, try less stringent conditions in the setting box.
If the sequence amplified doesn't include your sequence of interest – limit the
extra sequence before and after the amplified fragment.
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PCR program:
A basic PCR program that works very well (on which many modifications can be made)
is the following one –
PCR mix –
DNA – 50ng final
PCR buffer 10X no Mg++
Mg++ 25mM (to a final
concentration of 2mM)1
dNTPs 10X solution2
Primers3
Enzyme
ddH2O
Total
X1 (quantities in
microliters)
2 ul
2.5 ul
2 ul (1.5-1.75)
X____
2.5 ul
0.5F + 0.5R
25ul
PCR program –
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
950c 3min
940c 45sec
600c 45sec
720c 45sec
goto II X5
940c 45sec
550c 45sec
720c 45sec4
goto VI X 34
720c 10min
END
Calculating Tm for the annealing Temperature –
Calculate 20C for every A or T and 40C for every G or C.
Choose use the annealing temperature of the lower one of the primers as the annealing
temperature for step III in the PCR reaction and that temperature –(4-5)0C for the
annealing temperature in step VII of the PCR reaction.
If there is no product the temps at III and VII can be lowered in a couple of Degs.
1
Can use 2mM concentration of Mg++ for less stringent conditions or 1.5 or 1 for higher stringency =
reduce background.
2
Each dNTP is at 2mM concentration in the 10X solution and in the PCR the final concentration should be
200micromolar each.
3
In the PCR reaction should be in 0.2µM each, I use a 10µM stock solution.
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This step can be longer – the rule of thumb is 1 min per 1000 bp.
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