Team:DTU-Denmark/Notebook/20 August 2013

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20 August 2013

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Contents

lab 208

Main purpose

  • PCR to linearize pZA21::RFP::araBAD

Who was in the lab

Kristian, Helen, Henrike, Julia

Procedure

PCR to linearize pZA21::RFP::araBAD

  • Template: pZA21::RFP::araBAD
  • Primers: 55a and 55b
  • Program:
temperature time cycles
98C 2:00 -
98C 0:10 36
65C 1:00 36
72C 3:00 36
72C 5:00 -
10C hold -

The table shows the composition of each reaction:

component (per reaction) 1 2 3 4 5 6 Neg
dNTPs 1uL 1uL 1uL 1uL 1uL 1uL -
buffer (GC/HF + volume) HF + 10uL HF + 10uL HF + 10uL GC + 10uL HF + 10uL HF + 10uL HF + 10uL
X7 polymerase 0.5uL 0.5uL 0.5uL 0.5uL 0.5uL 0.5uL 0.5uL
MilliQ water 29uL 21.5uL 31.5uL 29uL 31.5uL 31.5uL 32.5uL
template 1uL 1uL 1uL 1uL 1uL 1uL -
FW primer 55a 3uL 3uL 3uL 3uL 3uL 3uL 3uL
RV primer 55b 3uL 3uL 3uL 3uL 3uL 3uL 3uL
DMSO (100%) 2.5uL (5%) - - 2.5uL (5%) - - -
Betaine (5M) - 10uL (1M) - - - - -

Results

Gels

loaded more samples from the screening PCR for BioBrick creation yesterday

  • 1 kb ladder
  • screening D12
  • screening D11
  • screening D10
  • screening D9
  • screening D8
  • screening D7
  • screening D6
  • screening D5
  • screening D4
  • screening D3
  • screening D2
  • screening D1
  • screening C12
  • screening C11
  • screening C10
  • screening C9
  • screening C8
  • 1 kb ladder

(pic big gel)

  • 1 kb ladder
  • screening C7
  • screening C6
  • screening C5
  • screening C4
  • screening C3
  • screening C2
  • screening C1
  • screening B12
  • screening B11
  • screening B10
  • screening B9
  • screening B8
  • screening B7
  • 1 kb ladder

(pic small gel)

purification gels

Pooled Nir2 and Nir1 samples that gave bands when Kristian made a test (to give the guy who is making polymerases).

  • 1 kb ladder
  • pZA21::RFP::AraBAD
  • pZA21::RFP::AraBAD
  • pZA21::RFP::AraBAD
  • Nir2
  • Nir2
  • Nir2
  • Nir1
  • Nir1
  • Nir1
  • Nir1
  • 1 kb ladder

Second purification

  • 1 kb ladder
  • pZA21::RFP::AraBAD, sample 1 (from todays PCR)
  • Nir1
  • Nir1
  • Nir2
  • Nir2
  • Nir2
  • 1 kb ladder

Conclusion

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