Melbourne/Lab Notebook Weeks 9-12

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Contents

Week 9

19 Aug 2007

  • Picked 3-4 transformants from "ligation" plates (17/8) and Grew up liquid cultures.
  • Transformed the two ligation reactions in the pink rack in the cold room and plated on Gen + Amp plate.


20 Aug 2007

Desired products:

    1. 450bp (95bp different from CI-2 used as control)
    2. 450bp
    3. 450bp
    4. 1kb
    5. 1kb
  • These were run on a gel.


OmpR-inverter
-Vector: OmpR (AmpR) miniprepped P1 15P from 10/7 digested with S/P.
-Insert: c1 inverter (1kb fragment) P2 13K A with X/P.


RBS-LacZ
-Vector: LacZ (KanR) P2 9E from 6/8 with E/X.
-Insert: RBS (GenR) P4 8J from 6/7 with E/S.


Digested for 2h and heat inactivated for 10min at 80degC.


21 Aug 2007

  • Picked 4 colonies from fresh ligation/transformation to Growing up.

- These transformations were made as follows:

    • Ligated
      1. 5ul of Purified insert from 17/8 (CI-2 E/S)
      2. 5ul of Redigested P1 5G with E/X and heat inactivated at 60degC for 10min.


22 Aug 2007

  • Miniprepped L1-L4 (liquid cultures from 21/8).
  • Digested these for ligations.
    1. L1 X/P
    2. L2 X/P
    3. L3 X/P
    4. L4 X/P
    5. CI-2 X/P (control for 350bp)
    6. P1 15P S/P

All L1-L4 had the 450bp desired band.

  • Gel purified 450bp band from L3 and a 950bp band from the P2 13K digest.

OmpR-L3 (RBS-LacZa-double term)
-Vector= P1 15P S/P (above)
-Insert= L3 X/P (above)

OmpR-c1 inverter (P2 13K)
-Vector= P1 15P S/P (above)
-Insert= P1 15P S/P (above)

Ligations were kept overnight at 4degC.


23 Aug 2007

  • Transformed ligations from 22/8 into competent NM522 cells.


24 Aug 2007

  • Liquid cultures were made of transformations from 23/8. 6 colonies were picked from each ligation.


25 Aug 2007

  • Ran on a Gel 16 samples that were miniprepped.
  • Gel purified 1000bp fragment from ompR-c1 inverter ligation digest and a 600bp fragment from ompR-L3 ligation digest.

Week 10

26 Aug 2007

(all from 25/8) Left overnight at 4degC.


27 Aug 2007

Plated on Amp plates.


28 Aug 2007

  • Picked six colonies from each plate (27/8) and cultured at 37degC for 13 hours.


29 Aug 2007

-The M30109 ligations yielded random fragments: something wrong with the part? - P1 15P-L3 and P1 15P-c1 inverter ligations had bands of the desired size.


30 Aug 2007

  • Set up minipreps from 29/8 for sequencing.

OmpR-L3 and OmpR-c1 inverter were the desired sequences.

Parts created:
OmpR-RBS-LacZa-double terminator

P115-P213K


31 Aug 2007


1 Sept 2007

Week 11

2 Sept 2007


3 Sept 2007


4 Sept 2007


5 Sept 2007


6 Sept 2007


7 Sept 2007


8 Sept 2007


Week 12

9 Sept 2007


10 Sept 2007


11 Sept 2007


12 Sept 2007


13 Sept 2007


14 Sept 2007


15 Sept 2007