Expand Long Template PCR

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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.
Wild type mix: (example)
Reagent
H2O
10 x GOLD PCR
Buffer
25 mM MgCl2
10 mM dNTP's
100 μM PrimerA
100 μM PrimerB
Ampli TAQ GOLD


Mutant mix: (example)
1x
18.9
14
264.6
2.5
2.5
0.5
0.2
0.2
0.2
35
35
7
2.8
2.8
2.8
350
25
Reagent
H2O
10 x GOLD PCR
Buffer
25 mM MgCl2
10 mM dNTP's
100 μM PrimerA
100 μM PrimerC
Ampli TAQ GOLD
1x
18.9
14
264.6
2.5
2.5
0.5
0.2
0.2
0.2
35
35
7
2.8
2.8
2.8
350
25
Set up reaction components in a sterile microfuge tube (on ice).
Aliquot 25μl of Mix into each PCR tube.
11/30/07
Erin Osborne




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
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