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MOLECULAR BIOLOGY: WORKING WITH DNA

POLYMERASE CHAIN REACTION

PCR FROM WHOLE YEAST

PCR from Whole Yeast
Contributor: The Laboratory of Andrew Murray at Harvard University
 
Overview
This protocol can be used to sequence from a plasmid, use 50-fold excess of Primer 1 (2 μM) vs. Primer 2 (0.04 μM) to obtain a mainly single stranded product, then use the sequencing Primer and sequence directly without purifying the product.
 
Procedure
1. Pick one yeast colony and resuspend it in 100 μl Reaction Mix in a 0.5 ml microcentrifuge tube.

2. Boil 5 min, centrifuge briefly and cool.

3. Add 0.5 μl Taq Polymerase, overlay each reaction tube with Mineral Oil

4. Amplify for 30 cycles:

94°C 1 min

42°C 2 min

65°C 4 min

Final extension at 65°C for 5 min

5. Run 10 to 15 μl on an Agarose gel (see protocol for Agarose Gel Electrophoresis of DNA).

Solutions
TE Buffer   10 mM Tris
pH 8.0
1 mM EDTA
Chloroform/Isoamyl Alcohol   25:1 Chloroform:Isoamyl Alcohol (CAUTION! see Hint #1)
Phenol/Chloroform   1:1 Phenol:Chloroform (CAUTION! see Hint #1)
Reaction Mix   5 μl 2 μM 3' Primer
63.5 μl ddH2O
10 μl 10X PCR Buffer
16 μl dNTP's (1.25 mM dATP, dCTP, dGTP, dTTP)
5 μl 2 μM 5' Primer
PCR Buffer (10X)   15 mM MgCl2
500 mM KCl
100 mM Tris, pH 8.3
 
BioReagents and Chemicals
Phenol
Ammonium Acetate
DNA Polymerase, Taq
EDTA
Ethanol
Oligonucleotide
Mineral Oil
Tris
Potassium Chloride
dCTP
dTTP
dGTP
dATP
Magnesium Chloride
Isoamyl Alcohol
Chloroform
 
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.