Holmes, Dawn 1-2
PCR of Environmental Genomic DNA
(This protocol must be done in a sterile hood)
1. Clean all surfaces in the hood including the work area, walls, tip boxes, tube boxes,
tube racks and pipits. Wipe down all surfaces using 10% bleach, DNA Away, and
then 70% ethanol.
2. Close hood and turn on the UV light for at least 20 minutes.
3. Remove reagents from -20oC freezer and thaw as described below.
In ice: 20pmole/ul primers, 10mM dNTP Mix, 100X BSA, Taq Polymerase (must
stay in ice)
In the UV cross linker: Q buffer, 10X buffer, MgCl2 buffer, sterile dH2O
4. In the sterile hood close one 1.5mL tube for each primer set and 0.2mL tubes for each
PCR reaction. Place all the closed tubes into the UV cross linker with the above
reagents not kept on ice and run the UV for 600 seconds.
5. To make a master mix for each primer set multiply the volumes in the recipe below
by the number of DNA samples that will be PCR amplified plus one reaction with no
DNA as the negative control.
Mix for PCR of Environmental Genomic DNA (50uL reaction)
Ingredient
Volume
16.75uL
Sterile dH2O
10uL
Q Buffer
5uL
10X Buffer
6uL
MgCl2 Buffer
10mM dNTP Mix (conc/dNTP) 1uL
2.5uL
20pmole/ul Forward Primer
2.5uL
20pmole/ul Reverse Primer
1uL
100X BSA
0.25uL
Taq Polymerase
6. If a master mix was made aliquot 45uL into each 0.2mL tube.
7. Add 5uL of the appropriate genomic environmental DNA template to each tube,
except for the negative control. For the negative control replace the DNA with 5uL
of sterile dH2O.
8. Close the tubes, place in a thermocycler, and run a typical PCR program like the one
below using the correct annealing temperature.
1. 95oC for 5 minutes
2. 95oC for 1 minute
3. Higher annealing temperature for 1.5 minutes
4. 72oC for 1.5 minutes
5. Go to step 2 for 19 cycles with a decrease in annealing temperature by
0.5oC for each cycle
6. 95oC for 1 minute
7. Lower annealing temperature for 1.5 minutes
8. 72oC for 1.5 minutes
9. Go to step 6 for 19 cycles
10. 72oC for 10 minutes
Holmes, Dawn 2-2
11. 4oC forever
Notes: Generally the higher annealing temp is about 5oC below the lowest melting
temperature for the primer set and the lower annealing temperature is 5oC below
higher annealing temperature of the primer set. If amplifying a longer gene the
cycling times will need to be increased relative to the amplicon length.
9. Remove from thermocycler and store at -20oC.