S. cerevisiae colony
PCR (Mark Hickman)
1.
Pick up with
a toothpick an entire
colony (preferably fresh) from YPD plate and place in the bottom of a
PCR
tube. Repeat for all samples.
Then microwave the tubes on high power
for 30 sec. (The first time a set
of primers are used, include a wild-type purified DNA control, if
possible. Add the DNA to reaction
mix in step 3Ñdo not autoclave.)
2.
Make up
enough reaction mixture for
all samples. For each 50 ul
reaction:
5 ul
10X
PCR buffer ÐMgCl2 (Invitrogen)
5 ul
10X
dNTPs (stock = 2mM each dNTP)
1.5 ul 50
mM
MgCl2 (Invitrogen)
0.5 ul primer
1
0.5 ul primer
2
0.25 ul Taq
(Invitrogen)
37.25 ul dH2O
3.
Resuspend
each colony in the
reaction mixture by pipeting up and down.
4.
PCR
conditions:
a.
Annealing
temp must be less than the
Tm of both oligos (a couple degrees lower than the lowest Tm seems to
work
better).
b.
Extension
time should be greater
than 1 min per kb of product (Round up to the next minute; this has
worked for
products up to 5 kb with an extension time of 5 min).