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PCR amplification
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PCR amplification

Introduction

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Materials

Procedure

  • Master mix preparation
  1. Master mix
Table1
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1
I.2.a. PCR mix preparation (48.75µL/tube x n tubes)Pour 40Pour 28
2
reactifsCfvol µL/reaction42.75
3
5X superfi1X1040028047.75
4
Primer Fw(P33) (10µM)0.5µM2.510070
5
Primer Rw(P34) (10µM)0.5µM2.510070
6
dNTPs200µM14029
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platinum DNA polymerase1X14029
8
H2031.751270920.75
8 rows
  1. Add 1.25 µL of DNA per tube
  1. Cycle of the PCR
Cycle for PCR guide
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1
98°C3min
2
98°C10sec
3
60°C10sec35X
4
72°C5 seconds elongation (30seconds per kb) also verify the optimal temperature for the polymerase
5
72°C5min
5 rows
cycle PCR target
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A
B
C
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1
98°C3min
2
98°C10sec
3
60°C10sec35X
4
72°C30 seconds elongation (30seconds per kb) also verify the optimal temperature for the polymerase
5
72°C5min
5 rows
  1. Run the PCR
  • Gel verification
  1. Use DEPC (Diethylpyrocarbonate) to clean tools and deactivate RNAse
  1. Agarose gel : 0.8 % in TAE 1X : weight the wanted amount of powder and add corresponding volume of TAE 0.5X (here 0.8 g for 100mL ), microwave for about 3 minutes until liquid is fully homogeneous and wait for it to cool down before usage
  1. colored with cyber safe or diamond die if RNA 2µL
  1. Loading: 1 µL loading dye + 4µL of DNA/RNA
  1. Run the gel 30 min at 100 V

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