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	[[Melbourne/OmpR-RBS-LacZa-double terminator|OmpR-RBS-LacZa-double terminator]]		[[Melbourne/OmpR-RBS-LacZa-double terminator|OmpR-RBS-LacZa-double terminator]]

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Revision as of 10:01, 9 October 2007

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Contents 1 Week 9 2 Week 10 3 Week 11 4 Week 12

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

• Miniprepped liquid cultures from 19/8 labeled LA, LB, LC and P2 13K A, P2 13K B.
• Concentration of DNA was measured to be:
  A- 221ng/ul
  B-224ng/ul.
  

• Digested:
  1. LA X/S
  2. LB X/S
  3. LC X/S
  4. P2 13K A X/S
  5. P2 13K B X/S

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.

• Digestions were set up for ligations.

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.

• Made Glycerol Stocks of P2 13K using dry ice method.

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.

• Recieved M30109 and CP919.

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

• The above were Ran on a Gel together with P2 13K X/P digest from 21/8.

All L1-L4 had the 450bp desired band.

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

• Ligated the following:

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

• Miniprepped cultures from 23/8 and M30109 cells.
• Digested:
  • 12 minipreps from ligations X/P
  • 2 minipreps from M30109 cells S/P
  • 2 minipreps from M30109 cells X/P

• 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

• Ligated
  1. M30109 S/P to ompR-L3 X/P
  2. M30109 S/P to ompR-c1 inv X/P

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

27 Aug 2007

• Transformed
  1. M30109 S/P to ompR-L3 X/P (26/8)
  2. M30109 S/P to ompR-c1 inv X/P (26/8)
  3. P1 15P-L3 (from 22/8)
  4. P1 15P-c1 inverter (from 22/8)

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

• Miniprepped 24 cultures from 29/8.
• Diagnosted all with X/S.
• Ran on a Gel

-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