IGEM:Arizona State/2014/Notebook/asu igem 2014/2014/08/04

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Preparing another RBS-TesA and LacI Ligation

  • Digested more of RBS-TesA with X and P(insert). Digested more of LacI with S and P (backbone).
TesA+RBS insert with LacI+backbone
Reagent RBS-TesA (μL) LacI (μL)
DNA 15.0 15.0
10X fast Digest Buffer 2.0 2.0
PstI 1.0 1.0
XbaI 1.0 .0
SpeI 0.0 1.0
H2O 1.0 1.0
37°C for 1 hour
  • Both samples used in the Clean and Concentrate kit
  • Treat the LacI with Antarctic Phsophotase.
Antarctic Phsophotase Treatment
Reagent
LacI DNA 10.0
Antarctic Phosphotase 1.0
Phosphotase Buffer 2.0
H2O 7.0
37°C for 1 hour then 65°C for 5 min
  • The RBS-TesA and LacI DNA samples were quantified:
  • RBS-TesA: 39.157 ng/μL
  • LacI: 62.579 ng/μL
  • Overnight ligation reactions were conducted for the test ligation and the negative control.
TesA+RBS insert with LacI+backbone
Reagent 1 (μL) Control (μL)
TesA+RBS 1.25 0.0
LacI 1.0 1.0
Ligation Buffer 1.0 1.0
Ligase 1.0 1.0
H2O 5.75 7.0
16°C overnight

ETHANOL PLASMID

  • Started seeds of 3 seperate colonies of ethanol plasmid
    • 50 ml LB w/ Chloramphenicol + picked colony
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