Team:TUDelft/13 August 2009

From 2009.igem.org

(Difference between revisions)
(→Calin)
(→Calin)
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===Calin===
===Calin===
-
Knockout day.
+
Knockout day. [http://openwetware.org/wiki/NanoBio:_Protocol_for_gene_knockout NanoBio: Protocol for gene knockout]
-
Made 1M l-arabinose stock.
+
Made 1M l-arabinose stock. 150mg into 0.85 mL ddH20.
Digested CE with EcoRI (10uL DNA).
Digested CE with EcoRI (10uL DNA).
Line 77: Line 77:
In 30C incubator at 13:25
In 30C incubator at 13:25
 +
 +
14:40 0.497 OD
 +
 +
on Ice at 14:57
 +
 +
centrifuge 1 @ 15:13 - 10 min 4000 rcf 3C
 +
 +
centrifuge 2 @ 15:44 - 10 min 4000 rcf 3C
 +
 +
centrifuge 3 @ 16:10 - 10 min 4000 rcf 3C
 +
 +
12 plates made, of which 8 are various controls
 +
 +
{| border="1" align="center"
 +
| Plate ID || Knockout || Variables || AB
 +
|- align="center"
 +
| 1H || oriTR_KO || +L-arabinose +PCR  || 1xKAN
 +
|- align="center"
 +
| 1L || oriTR_KO || +L-arabinose +PCR || 0.5xKAN
 +
|- align="center"
 +
| 2 || oriTR_KO || +L-arabinose -PCR || TRI
 +
|- align="center"
 +
| 3H || oriTR_KO || -L-arabinose +PCR || 1xKAN
 +
|- align="center"
 +
| 3L || oriTR_KO || -L-arabinose +PCR || 0.5xKAN
 +
|- align="center"
 +
| 4 || oriTR_KO || -L-arabinose -PCR || TRI
 +
|- align="center"
 +
| 5H || trbK_KO || +L-arabinose +PCR || 1xKAN
 +
|- align="center"
 +
| 5L || trbK_KO || +L-arabinose +PCR || 0.5xKAN
 +
|- align="center"
 +
| 6 || trbK_KO || +L-arabinose -PCR  || TRI
 +
|- align="center"
 +
| 7H || trbK_KO || -L-arabinose +PCR || 1xKAN
 +
|- align="center"
 +
| 7L || trbK_KO || -L-arabinose +PCR || 0.5xKAN
 +
|- align="center"
 +
| 8 || trbK_KO || -L-arabinose -PCR || TRI
 +
|}
 +
 +
Made 5mL tube cultures for assemblies CB and CC.
{{Template:TUDelftiGEM2009_end}}
{{Template:TUDelftiGEM2009_end}}

Revision as of 17:00, 13 August 2009

Lab Notebook

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13 August 2009

Calin

Knockout day. [http://openwetware.org/wiki/NanoBio:_Protocol_for_gene_knockout NanoBio: Protocol for gene knockout]

Made 1M l-arabinose stock. 150mg into 0.85 mL ddH20.

Digested CE with EcoRI (10uL DNA).

Yesterdays gel:

Gel120809-delay-conj.png


Well Part Expected Plasmid Size Status
1 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ✔
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17 trbK X+P ✔
18 GFP-gen E+S ✔
19 pTet+RBS E+S  ?
20 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] ✔

Made 12 plates. 4 High KAN. 4 Low KAN. 4 TRI.

Placed 50uL of overnight R751 + pKD46 culture into 4 x 5 mL tubes.

In 30C incubator at 11:15

11:45 0.034 OD

12:30 0.065 OD

13:17 0.126 OD

Induced +L-arabinose tubes with L-arabinose (40uL 1M stock added to 4mL). Control tube has no L-arabinose added.

In 30C incubator at 13:25

14:40 0.497 OD

on Ice at 14:57

centrifuge 1 @ 15:13 - 10 min 4000 rcf 3C

centrifuge 2 @ 15:44 - 10 min 4000 rcf 3C

centrifuge 3 @ 16:10 - 10 min 4000 rcf 3C

12 plates made, of which 8 are various controls

Plate ID Knockout Variables AB
1H oriTR_KO +L-arabinose +PCR 1xKAN
1L oriTR_KO +L-arabinose +PCR 0.5xKAN
2 oriTR_KO +L-arabinose -PCR TRI
3H oriTR_KO -L-arabinose +PCR 1xKAN
3L oriTR_KO -L-arabinose +PCR 0.5xKAN
4 oriTR_KO -L-arabinose -PCR TRI
5H trbK_KO +L-arabinose +PCR 1xKAN
5L trbK_KO +L-arabinose +PCR 0.5xKAN
6 trbK_KO +L-arabinose -PCR TRI
7H trbK_KO -L-arabinose +PCR 1xKAN
7L trbK_KO -L-arabinose +PCR 0.5xKAN
8 trbK_KO -L-arabinose -PCR TRI

Made 5mL tube cultures for assemblies CB and CC.

Retrieved from "http://2009.igem.org/Team:TUDelft/13_August_2009"