Team:DTU-Denmark/Notebook/29 August 2013

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29 August 2013

Contents

lab 208


Main purpose


Who was in the lab


Procedure


Introduce endings for USER cloning in pSB1C3

In order to introduce our constructs in the biobrick vector we need to introduce special endings that are necessary for the USER cloning reaction. The table shows the composition of each reaction:

component 1 2 3 Neg
dNTPs 1uL 1uL 1uL 1uL
HF buffer 10uL 10uL 10uL 10uL
X7 polymerase 0.5uL 0.5uL 0.5uL 0.5uL
MilliQ water 28.5uL 28.5uL 28.5uL 29.5uL
template pSB1C3 1uL 1uL 1uL -
FW primer 57a2 3uL 3uL 3uL 3uL
RV primer 57b 3uL 3uL 3uL 3uL
DMSO (100%) 2uL (4%) 2uL (4%) 2uL (4%) 2uL (4%)
MgCl2 (50mM) 1uL (1mM) 1uL (1mM) 1uL (1mM) 1uL (1mM)

Results


Gel pictures

Ran gels on yesterday's PCRs to create psB1C3 with USER endings.

Small gel:

  • 1 kb ladder
  • 2%, pur
  • 4%, pur
  • 5%, pur
  • 4%, 1uL, pur
  • 4%, 2uL, pur
  • 1M, pur
  • 2%, HQ
  • 4%, HQ
  • 5%, HQ
  • 4%, 1uL, HQ
  • 4%, 2uL, HQ
  • 1M HQ
  • 1 kb ladder (ladder was almost empty so I put another one in the next lane)
  • 1 kb ladder

big gel (samples from touch down PCR):

  • 1 kb ladder
  • GC, HQ
  • 2% HQ
  • 4% HQ
  • 5% HQ
  • 4%, 1uL, HQ
  • 4%, 2uL, HQ
  • 1M HQ
  • GC pur
  • 2%, pur
  • 4%, pur
  • 5%, pur
  • 4%, 1uL, pur
  • 4%, 2uL, pur
  • 1M pur
  • neg
  • 1 kb ladder

Conclusion


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