Team:TUDelft/12 August 2009

From 2009.igem.org

(Difference between revisions)
Line 45: Line 45:
DNA:
DNA:
 +
{| border="1" align="center"
 +
| Biobrick || DNA concentration (ng/uL)
 +
|- align="center"
 +
| E0240 || 72.2
 +
|- align="center"
 +
| B0031 || 56.2
 +
|- align="center"
 +
| J04630 || 130.9
 +
|- align="center"
 +
| J13002 || 34.7
 +
|- align="center"
 +
| I13507 || 105.4
 +
|- align="center"
 +
| R0010 || 31.1
 +
|- align="center"
 +
| R0040 || 27
 +
|- align="center"
 +
| pSB1C3 || 68.6
 +
|- align="center"
 +
| pSB1A3 || 85.1
 +
|- align="center"
 +
| CE || 27.5
 +
|- align="center"
 +
| CB || 6.4
 +
|}
   
   
-
 
{{Template:TUDelftiGEM2009_end}}
{{Template:TUDelftiGEM2009_end}}

Revision as of 14:39, 29 August 2009

Lab Notebook

July
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
 

12 August 2009

Calin

No growth for assembly CB. CE has many colonies. pKD plasmids all have growth, plates put into fridge.

Did a digest on trbK with X+P (4 uL DNA). Disest on GFP-gen with E+S (5 uL DNA). Digest on pTet-RBS E+S (8 uL DNA). Did ligations CB (GFPgen+oriTR on pSB1C3) and CC (pTet-RBS+trbK on pSB1C3).

Rehydrated knockout primers to make 100uM stock. Made the linear fragments needed for the knockout. Made 10uM primer mix for both oriT_KO_PCR and trbK_KO_PCR.

Made master mix for Pfx platinum PCR.

Ran both oriT_KO_PCR and trbK_KO_PCR PCR reactions using pKD4 plasmid as template.

Did a PCR purify. On the trbK_KO_PCR sample the pH was too high. Added sodium acetate and a tiny bit of acetic acid to lower pH. Did a PCR purify using the Qiaquick kit.

Checked concentrations:

oriT-R_KO_PCR 80 1.88 1.93

trbK_KO_PCR 41.6 1.79 1.14

Ran a DpnI digestion on both. Enzyme used is old. Still working?

Ran another PCR purify.

Checked concentrations:

oriT-R_KO_PCR 72.8 1.93 1.78

trbK_KO_PCR 34.5 1.70 1.17

Sriram transformed CB and CC with heat shock on CAM plates.

Made another TRI+AMP plate with pKD46 and R751 for the knockout. Also made a 5mL tube culture.

Daniel

Today I did miniprep on the tube cultures which all had growth. Besides I did glycerol stocks for future generations of TUDelft's iGEM teams. After miniprep, I checked the DNA's concentration (which somehow I always got low concentrations compared with the other members, you will see).

DNA:

Biobrick DNA concentration (ng/uL)
E0240 72.2
B0031 56.2
J04630 130.9
J13002 34.7
I13507 105.4
R0010 31.1
R0040 27
pSB1C3 68.6
pSB1A3 85.1
CE 27.5
CB 6.4