Holmes, Dawn 1-1
PCR of Pure Culture Genomic DNA
(If using highly specific primers- PCR can be set up on bench top.)
(If the reagents are used on the bench top they cannot be used in the hood later)
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 Pure Culture Genomic DNA (50uL reaction)
Ingredient
Volume
19.75uL
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 48uL into each 0.2mL tube.
7. Add 2uL of the appropriate DNA template to each tube, except for the negative
control. For the negative control replace the DNA with 2uL of sterile dH2O.
8. Close the tubes, place in a thermocycler, and run the following PCR program.
1. 95oC for 5 minutes
2. 95oC for 30 seconds
3. Annealing Temperature for 45 seconds
4. 72oC for 45 seconds
5. Go to step 2 for 34 cycles
6. 72oC for 10 minutes
7. 4oC forever
Note: The annealing temperature is generally determined by checking the melting
temperature of each primer in the set and then set at about 5oC below the lower
melting temperature. If amplifying a longer gene the cycling times will need to be
increased relative to the amplicon length. Generally increasing the cycling times to
45 seconds, 1 minute, and 1 minute is adequate for 1000 base pairs.
9. Remove reactions from thermocycler and store at -20oC.