TGGE Test Kit protocol

TGGE Test Kit protocol
TGGE Testkit (024-050)
1 Introduction
The TGGE test kit was developed to get familiar with the TGGE system. It contains 3 tubes
with different DNA samples and 1 tube with loading buffer.
wild type DNA
homoduplex
40 µl
mutant DNA
homoduplex
40 µl
mutant / wildtype DNA
heteroduplex
400 µl
loading buffer
1 ml
Prior to use, samples should to be diluted as described below.
Total sample volume per slot:
Maxi System (024-200)
Standard “Mini” System (024-000)
perpendicular TGGE
200 µl
50 µl
parallel TGGE
5 µl
5 µl
buffer system
1 x TAE (Sambrook et al. 1989)
1 x TAE (Sambrook et al. 1989)
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2 Protocol for TGGE standard “Mini” system (024-000)
•
Running buffer: 1 x TAE
•
Gel composition: 8% Polyacrylamid, 8 M Urea, 1 x TAE, 2% Glycerol.
•
For preparation of solutions and casting of gels please refer to the TGGE system
manual.
TGGE standard „Mini“ system (024-000)
sample
preparation
perpendicular TGGE
Parallel TGGE
25 µl sample (heteroduplex)
2,5 µl sample (wild type, mutant or
heteroduplex)
25 µl loading buffer
2,5 µl loading buffer
Vortex, quick spin and load 50 µl in Vortex, quick spin and load 5µl each per
the broad slot
slot
pre run
main run
temperature T1: 20°C,
T1: 20°C
temperature T2: 20°C
T2: 20°C
voltage 250 V
Voltage 250V
time 7 min
time 7 min
temperature T1: 30°C,
T1: 39°C
temperature T2: 70°C
T2: 54°C
voltage 250 V
Voltage 250V
time 40 min
time 50 min
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3 Protocol for TGGE Maxi system (024-200)
•
Running buffer 1 x TAE
•
Gel composition: 8% PAA gel with 8 M Urea , 2% Glycerol, 1 x TAE
•
For preparation of gel solutions and buffers see TGGE maxi user manual
TGGE MAXI system (024-200)
Sample
preparation
perpendicular TGGE
parallel TGGE
100 µl sample (heteroduplex)
2,5 µl sample (wild type, mutant or
heteroduplex)
100 µl loading buffer
2,5 µl loading buffer
pre run
Vortex, quick spin and load 200 µl
in the broad slot
Vortex, quick spin and load 5µl each per
slot
temperature T1: 20°C,
T1: 20°C
temperature T2: 20°C
T2: 20°C
voltage 300 V
voltage 300V
time 10 min
time 10 min
temperature T1: 30°C,
Gradient
equilibration temperature T2: 70°C
Main run
T1: 39°C
T2: 54°C
voltage 0V
voltage 0V
time 10 min
time 10 min
temperature T1: 30°C,
T1: 39°C
temperature T2: 70°C
T2: 54°C
voltage 300V
voltage 300V
time 3,5 h
time 4h
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4 Gel examples
perpendicular TGGE
M
HD
M
= Marker
Wt
= Wild type
Mt
= Mutant
Hd
= Heteroduplex
parallel TGGE
M
M Mt Wt HD Mt Wt HD M
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