Team:Paris Saclay/Notebook/July/30

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(C - Lemon scent)
(PCR of BBa_K762100)
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'''Protocol'''
'''Protocol'''
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Add into a PCR tube the following:
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Add into each PCR tube the following:
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{| class="wikitable centre" width="50%"

Revision as of 14:57, 30 July 2014

Contents

Wednesday 30th July

Lab Work

C - Lemon scent

PCR of BBa_K762100

by Sean

This was a second attempt at yesterday's PCR, with several changes in parameters. Two tubes were prepared: one with yesterday's enzyme and another with a newer version of the same enzyme in order to determine if yesterday's enzyme was one of the causes of the rather unsatisfactory results.

Oligonucleotides used: iPS66, iPS67

Oligonucleotides were diluted twice from 100µM to 50µM.

Protocol

Add into each PCR tube the following:

Component For a total volume of 50μl
H2O 35.5μl
Phusion buffer 5X 10μl
dNTPs 1μl
iPS66 1μl
iPS67 1μl
BBa_K762100 1μl
Phusion DNA polymerase 0.25μl

Tube was placed in PCR machine with the following parameters.

Cycle step Temperature Time Cycle

Initial denaturation

98°C

1 min

1

Denaturation 98°C 15 s 25 - 30
Annealing variable 55°C 25 - 30
Extension 72°C 45 s 25-30
Final extension 72°C 10 min 1
Final extension 8°C hold 1

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