Team:TUDelft/13 August 2009

=13 August 2009=

Sriram
Today i understood that running the gel with ligated products is unnecessary which is clear in the below gel. I must not do this mistake again. I plated the transformed colonies on the plate.

Calin
Knockout day. NanoBio: Protocol for gene knockout

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

Digested CE with EcoRI (10uL DNA).

Yesterdays gel:



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

Tim Vos helped with electroporation step. Electroporator was set to 600 ohm.

After one hour 200 uL was plated. oriTR_KO +L-ara +PCR and trbK_KO +L-ara +PCR placed in fridge at 18:30.

Made 5mL tube cultures for assemblies CB and CC.

Todays 8 well gel to check the linear fragments for the knockout:



Daniel
Yesterday´s gel confirm the sizes of all the biobricks needed (see the wells 4-10 in the first gel of this site), I did a second round of digestions though, in order to have enough DNA. After a new gel and check sizes, I did the assemblies 1A and 2A (see locks and keys section). Also, I transformed by heat shock and electroporation these assemblies and did plates and culture tubes with respective antibiotic.