Team:Paris Bettencourt/Notebook/Riboswitch

July 27th

Goal

E coli DH5-alpha transformation with RFP from iGEM plate 4 - 2H (pSB1A3 backbone)

Procedure

  1. I took iGEM plate 4 - 2H (RFP, BBa_J04450 in PSB1A3 backbone) and resuspend it in 10 uL of distilled water.
  2. Used E coli competent cells from NEB 2 tubes c2987 (50uL) : 1 control (C) and one for transformation (T)
  3. Introduced 2 uL (200-300pg/ul) of my suspended plasmid solution in T.
  4. Waiting 30 min in ice
  5. Heat shock 30 sec 42°C
  6. Then 5min in ice
  7. Add 200uL SOC medium
  8. Recovery 2 hours then plate it on 3 different 100ug/mL Ampicillin LB
  9. Incubate 37°C till tomorrow

Results

The day after I had colonies growing, nothing on my control
    I took 2 different colonies and do a 8 hours culture in two different tubes T1 an T2 in LB + Amp 100ug/mL, in order to do a glycerol stock.


July 29th

Goal

Making chemically competent cells

Procedure

I followed the following method
  • The day before, start an overnight Liquid Culture of the E. coli strain. (5mL of LB + Antibiotics + Single colony)
  • The next day, set the Centrifuge at 4°C and place on ice a tube containing 15mL of 100mM CaCl2 and a tube containing 4mL of 100mM CaCl2 + 15% Glycerol.
  • In an Erlenmeyer, add 50mL of LB and 250uL of the Liquid Culture, (for quicker growth, use a larger Erlenmeyer)
  • Put the Erlenmeyer to shake at 37°C until the OD(600) reaches 0.4 - 0.5.
  • When the OD(600) is reached, transfer in a 50mL Falcon tube and centrifuge 10min at 4°C.
  • Remove the supernatant and resuspend in 15mL of ice cold 100mM CaCl2. -Leave on ice for 4h.
  • Centrifuge 10min at 4°C.
  • Remove the supernatant and resuspend in 4mL of ice cold 100mM CaCl2 + 15% Glycerol.
  • Aliquot in 1.5mL eppendorf tubes and store at -80°C until use.


    • July 30th

    Goal 1

    PCR my G block and backbone

    Procedure

  • PCR1 = Backbone + 068 and 069 primers, before purify
  • PCR2 = FINAL + 068 and 069 primers, before purify
  • Then PCR purification
  • Nanodrop : PCR 1 = 61ng/uL ; PCR2 = 25,4 ng/uL

    • Goal 2

    Digestion/Ligation/Transformation

    Procedure

  • PCR1 ADN : 40uL = 2,4 ug 4uL EcoRI, PstI, FastAp 12 uL FD buffer 56 uL water TOT =120uL
  • PCR2 ADN : 40uL = 1ug 6 uL EcoRI, PstI 12 uL FD buffer 64 uL water TOT= 128uL
  • 10 min at 37°C
  • Then PCR purification : Nanodrop : nanodrop : PCR 1 = 20,7 ng/uL ;PCR2 = 25,7