Melbourne/Lab Notebook Weeks 9-12
From 2007.igem.org
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*The above were [[Melbourne/Loading a DNA gel|Ran on a Gel]] together with [[Melbourne/BBa_Q04510|P2 13K]] X/P digest from 21/8. | *The above were [[Melbourne/Loading a DNA gel|Ran on a Gel]] together with [[Melbourne/BBa_Q04510|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 [[Melbourne/BBa_Q04510|P2 13K]] digest. | *Gel purified 450bp band from L3 and a 950bp band from the [[Melbourne/BBa_Q04510|P2 13K]] digest. | ||
*[[Melbourne/Ligation Protocol|Ligated]] the following: | *[[Melbourne/Ligation Protocol|Ligated]] the following: | ||
| + | <font color=crimson> OmpR-L3 (RBS-LacZa-double term)</font><BR> | ||
| + | -Vector= [[Melbourne/BBa_R0082|P1 15P]] S/P (above)<BR> | ||
| + | -Insert= L3 X/P (above) | ||
| + | |||
| + | <font color=crimson> OmpR-c1 incerter ([[Melbourne/BBa_Q04510|P2 13K]]) </font><BR> | ||
| + | -Vector= [[Melbourne/BBa_R0082|P1 15P]] S/P (above)<BR> | ||
| + | -Insert= [[Melbourne/BBa_R0082|P1 15P]] S/P (above) | ||
| + | |||
| + | Ligations were kept overnight at 4degC. | ||
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</font><BR> | </font><BR> | ||
| - | + | *[[Melbourne/Transformation Protocol|Transformed]] ligations from 22/8 into competent NM522 cells. | |
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</b> | </b> | ||
</font><BR> | </font><BR> | ||
| + | |||
| + | *[[Melbourne/Growing up cells|Liquid cultures]] were made of transformations from 23/8. Four colonies were picked from each ligation. | ||
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</b> | </b> | ||
</font><BR> | </font><BR> | ||
| + | |||
| + | *[[Melbourne/Miniprep protocol|Miniprepped]] cultures from 23/8. | ||
==Week 10== | ==Week 10== | ||
Revision as of 09:09, 9 October 2007
<Return to Lab notebook> <team home page>
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
- 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.
Desired products:
- 450bp (95bp different from CI-2 used as control)
- 450bp
- 450bp
- 1kb
- 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:
22 Aug 2007
- Miniprepped L1-L4 (liquid cultures from 21/8).
- Digested these for ligations.
- L1 X/P
- L2 X/P
- L3 X/P
- L4 X/P
- CI-2 X/P (control for 350bp)
- 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 incerter (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. Four colonies were picked from each ligation.
25 Aug 2007
- Miniprepped cultures from 23/8.
Week 10
26 Aug 2007
27 Aug 2007
28 Aug 2007
29 Aug 2007
30 Aug 2007
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
