Setting
up a PCR
Before gathering
any
materials or reagents determine the volumes of each reagent that will
be
needed. Fill out PCR details sheet.
Procedure
1.
Get a
container of ice and a PCR rack.
2.
Defrost
all reagents except Taq. Keep Taq on ice at all times.
PCR grade water
10 X Buffer
dNTP mix
forward primer @
5pm/µl
reverse primer @ 5pm/µl
Magnesium (MgCl2)
(if
necessary)
3.
Briefly
vortex each of the reagents except Taq and water. This is to ensure mixing
in case of settling during
freeze/thaw. Put all reagents on ice with Taq.
4.
Label 0.2
ml reaction tubes and 0.5 ml tube for
cocktail mix.
5.
Add
reagents to cocktail mix tube one by one in the
order listed above. Use volumes
previously determined and written on your PCR lab sheet.
Check off as each reagent is added to
the cocktail.
6.
Vortex to
mix thoroughly. (5 seconds @ maximum
speed). Put on ice.
7.
Add Taq
to cocktail mix and vortex gently to mix with
other reagents.
8.
Dispense
cocktail to reaction tubes. Work carefully
but quickly.
9.
Add DNA
or water (water replaces DNA in negative
controls) to reaction tubes.
10.
Make sure
that the lid of each reaction tube is
snapped shut (sealed) or the reaction will evaporate. Flick each tube
gently to
ensure mixing and that the contents are at the bottom of the tube.
11.
Place
reaction tubes in PCR machine and start
program.
12.
Put all
reagents away in -20°C freezer.
PCR program
1.
94° C for 2.5 min
2.
94° C for 30 s
3.
52-62° C for 45 s (depending on primer Tm)
4.
72° C for 1 – 4 min, depending on length of
product (1
min/kb)
5.
72° C for 10 min
6.
Hold at 4° C
Materials necessary
0.2
ml PCR tubes (individual or 8-strip)
0.5
ml microcentrifuge tube
Forward
and reverse primers @ 5 pM
10X
PCR Buffer
10
and 20 µl pipette tips
PCR
grade water
Ice
Equipment necessary
PCR
thermocycler
10 µl pipette
20 µl pipette
200 µl pipette