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September/3 September 2009
From 2009.igem.org
1. Colony check of cultures incubated on 9/1 and 9/2:
9/1: (5), (9), (18), (20), (21) all failed to produce cultures.
9/2: (9), (20), (21) failed to produce cultures.
The following plates produced cultures:
non-NB NB [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0080 1-14L] 20 200 [http://partsregistry.org/wiki/index.php?title=Part:BBa_I763011 1-15L] 6 5 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K117008 2-14L] 5 100 [http://partsregistry.org/wiki/index.php?title=Part:BBa_Q01511 1-16L] 7 10 [http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450 1-1K] 15 20 [http://partsregistry.org/wiki/index.php?title=Part:BBa_I763007 1-15J] 4 10 [http://partsregistry.org/wiki/index.php?title=Part:BBa_I0500 1-14N] 0 1 (NB=Norad Blue, a super-competent E.coli strain)
The NB cultures that produced colonies were inoculated into culture solution and incubated overnight.
2. Miniprep of the solution culture made from the only (21) colony produced so far.
3. Nanodrop check of the above miniprepped DNA as well as parts miniprepped yesterday ( (14)-A, (19)-A, (18)-A, (18)-B, (18)-C ).
result sample const. No.21 41.4 ng/uL 14-A 5.5 ng/uL 19-A 7.6 ng/uL 18-A 7.3 ng/uL 18-B 8.4 ng/uL 18-C 12.9 mg/uL
4. Ligation check (cut check: restriction digest + electrophoresis) of the above Nanodrop-checked plasmids.
No.21 , 18-A,B,C , 14 , 19 , ladder No.21 , 14 , 19 : ligation OK!
5. Transformation of parts ligated yesterday: (5), (9), (18), (20), (22), (23), (25), (26).
6. Restriction digest for ligation: vector-insert pairs for parts (9), (18), (24), (27).
Restriction enzyme-added DNA was reacted overnight at 37 degrees Celsius, for purification by electrophoresis + ligation tomorrow.
K204009
| Vector | Insert | ||
|---|---|---|---|
| 1-23L | 8 | No.4 | 8 |
| EcoRI | 1 | EcoRI | 1 |
| XbaI | 1 | SpeI | 1 |
| No.2 | 2 | No.2 | 2 |
| dH2O | 8 | dH2O | 8 |
| total | 20uL | total | 20uL |
K204024
| Vector | Insert | ||
|---|---|---|---|
| 1-23L | 8 | No.14 | 13 |
| EcoRI | 1 | EcoRI | 1 |
| XbaI | 1 | SpeI | 1 |
| No.2 | 2 | No.2 | 2 |
| dH2O | 8 | dH2O | 3 |
| total | 20uL | total | 20uL |
K204018
| Vector | Insert | ||
|---|---|---|---|
| 1-6I | 8 | No.10 | 8 |
| SpeI | 1 | XbaI | 1 |
| PstI | 1 | PstI | 1 |
| No.2 Buffer | 2 | No.2 | 2 |
| dH2O | 8 | dH2O | 8 |
| total | 20uL | total | 20uL |
K204027
| Vector | Insert | ||
|---|---|---|---|
| No.19 | 8 | 1-6I | 8 |
| SpeI | 1 | XbaI | 1 |
| PstI | 1 | PstI | 1 |
| No.2 Buffer | 2 | No.2 | 2 |
| dH2O | 8 | dH2O | 8 |
| total | 20uL | total | 20uL |
↓
37°C, O/N
