Yeast Colony PCR
Purpose:
This is a diagnostic PCR protocol which can distinguish wild-type from mutant colonies of yeast.
References:
Amplitaq GOLD Polymerase Hand-Out
Brandon Davies
Primers:
Protocol:
· Prepare the yeast. Set up 8-well PCR tubes with 20μl of YPD in each tube. To each tube, add a small toothpick-prick of each yeast to be tested. Be sure to include tubes for controls. You should use…
1. A wild-type control
2. A mutant control (if available)
3. A no yeast control
· Set up the master mixes of PCR. Make enough mix to amplify each yeast using both wild-type & mutant primers.
11/30/07
Erin Osborne
Wild type mix: (example)
Reagent / 1x / 14H2O / 18.9 / 264.6
10 x GOLD PCR Buffer / 2.5 / 35
25 mM MgCl2 / 2.5 / 35
10 mM dNTP's / 0.5 / 7
100 μM PrimerA / 0.2 / 2.8
100 μM PrimerB / 0.2 / 2.8
Ampli TAQ GOLD / 0.2 / 2.8
350
25
Mutant mix: (example)
Reagent / 1x / 14H2O / 18.9 / 264.6
10 x GOLD PCR Buffer / 2.5 / 35
25 mM MgCl2 / 2.5 / 35
10 mM dNTP's / 0.5 / 7
100 μM PrimerA / 0.2 / 2.8
100 μM PrimerC / 0.2 / 2.8
Ampli TAQ GOLD / 0.2 / 2.8
350
25
11/30/07
Erin Osborne
· Set up reaction components in a sterile microfuge tube (on ice).
· Aliquot 25μl of Mix into each PCR tube.
· Using a Multi-Channel Pipet, add 1μl of yeast cultures to each PCR tube. Cap tubes.
· Run the Thermocycle Profile
1. 94C 13:00
2. 94C 1:00
3. 50C 0:30
4. 72C 2:00***
5. Goto Step2 34x
6. 72C 10:00
7. 4C for ever
8. END
*** This time changes as your expected product length changes. Elongation time = 1min/1kb fragment + 1 min
· Add tubes to the thermocycler.
· To verify the products, run 10μl of each reaction on a 1 – 1.5% agarose gel + EtBr.
11/30/07
Erin Osborne