Sheffield Molecular Genetics Facility
Setting up a PCR reaction
1) Clean your area, pipettes etc with 10% bleach (can use UV if available)
2) Add the reagents to a 1.5ml tube. The values in the boxes- next page!-are
calculated for 1 sample. Multiply by the no. of PCR reactions you want to do +
10% to allow for pipetting inaccuracies. The calculations for most of the different
MgCl2 concentrations are given on the next page for reference.
3) If you are testing a lot of primers at the same MgCl2 concentration then make up a
big “mastermix” for all samples without the primer. Then split this into “submastermixes” before adding the primer.
4) Once the mastermix is prepared you can aliquot your DNA into wells in a
microtitre plate
5) Add 9ul of the mastermix to each DNA sample
6) Add a drop of oil, cover in Saranwrap and place in the PCR machine on the
correct temperature profile
For the demonstration we are going to use two sets of primers to sex our bird DNA samples
P2/P8 (Griffiths et al. 1998) and 2550F/2718R (Fridolfsson and Ellegren 1999)
(see The SMGF Bird sex typing database,
http://www.shef.ac.uk/misc/groups/molecol/smgfbirdsexing.html)
Both sets can be used at 2mM MgCl2 for most species
The PCR profiles are as below
Sheffield Molecular Genetics Facility
For demonstration we are going to use two sets of primers to sex our bird DNA
samples
P2/P8 (Griffiths et al. 1998) and 2550F/2718R (Fridolfsson and Ellegren 1999)
(see The SMGF Bird sex typing database,
http://www.shef.ac.uk/misc/groups/molecol/smgfbirdsexing.html)
Both sets can be used at 2mM MgCl2 for most species
The PCR profiles are as below
P2/P8: (visualised on DNA Sequencer)
1 cycle of 94- 2mins
then 40 cycles of:
94- 15 seconds
50- 20 seconds
72- 25 seconds
then 4C soak
2550F/2718R: ‘Touchdown’ from 60-50 (visualised on 2% agarose gel)
1 cycle of 94- 2mins
then 1 cycle of:
94- 30 seconds
60- 30 seconds
72- 30 seconds
Then the annealing temp. is dropped by 1 degree until 50 degrees is reached where 35
cycles are performed.
then 4C soak
References
Fridolfsson AK and Ellegren H (1999) A simple and universal method for molecular
sexing of non-ratite birds. Journal of Avian Biology, 30, 116-121.
Griffiths R, Double MC, Orr K and Dawson RJG (1998) A DNA test to sex most
birds. Molecular Ecology, 7, 1071-1075.
Sheffield Molecular Genetics Facility
Sheffield Molecular Genetics Facility
PCR reaction recipes
all volumes in ul
0.625 mM MgCl2
10x react. buffer IV
autolclaved water
25mM MgCl2
2mM dNTP’s
1.0
Taq (thermoprime plus)
Forward primer
Reverse primer
DNA
1.0
4.7
0.25
Total
10
0.05
1.0
1.0
1.0
…………………………………
1.0 mM MgCl2
10x react. buffer IV
autoclaved water
25mM MgCl2
2mM dNTP’s
1.0
Taq (thermoprime plus)
Forward primer
Reverse primer
DNA
1.0
4.55
0.4
Total
10
0.05
1.0
1.0
1.0
…………………………………
1.5 mM MgCl2
10x react. buffer IV
autoclaved water
25mM MgCl2
2mM dNTP’s
1.0
Taq (thermoprime plus)
Forward primer
Reverse primer
DNA
1.0
4.35
0.6
Total
10
0.05
1.0
1.0
1.0
…………………………………
Sheffield Molecular Genetics Facility
Sheffield Molecular Genetics Facility
PCR reaction recipes
2.0 mM MgCl2
10x react. buffer IV
autoclaved water
25mM MgCl2
2mM dNTP’s
1.0
Taq (thermoprime plus)
Forward primer
Reverse primer
DNA
1.0
4.15
0.8
Total
10
0.05
1.0
1.0
1.0
2.5 mM MgCl2
10x react. buffer IV
autoclaved water
25mM MgCl2
2mM dNTP’s
1.0
Taq (thermoprime plus)
Forward primer
Reverse primer
DNA
1.0
3.95
1.0
Total
10.5
0.05
1.0
1.0
1.0
2.5 mM MgCl2
10x react. buffer IV
water
25mM MgCl2
2mM dNTP’s
Taq (thermoprime plus)
Forward primer
Reverse primer
DNA
1.0
3.95
1.0
1.0
0.05
1.0
1.0
1.0
Total
10.5
Sheffield Molecular Genetics Facility