Team:TUDelft/21 July 2009

=21st July=

Sriram
Finally transformation worked in 5 plates [BBa_E0040 - GFP, BBa_C0040 - TetR, BBa_C0051 - cl, BBa_I12006 - λp, BBa_E1010 - mRFP1] out of 6 and for unknown reasons the biobrick BBa_I14018 - pBla was not transformed and not seen in the plate. At 5:45 PM the best colony from 5 plates were cultured in 5 ml culture tubes with respective antibiotics (two 1xKan and 3 1xAmp) and incubated at 37 &deg;C and 160 rpm. So tomorrow we can proceed to the DNA extraction step, since for assembly we can use pTet instead of pBla or any other constitutive promoter in future.

Found that the competent cells must be prepared. Tim Weenink has already cultured top 10 cells for competent cell preparation. I diluted the culture in 100ml flask and grown till 0.49 OD at 600nm. While the culture was growing I prepared the TSS buffer and filter sterilized with 0.2 µm syringe filter and stored in freezer. Then 50ml culture was split into two falcon tubes and centrifuged. After throwing the supernatant it was resuspended in each tube with 2.5ml TSS buffer.

Calin
Got transformed colonies in all 4 plates [BBa_I714031 - OriT-R, BBa_J23100 - strong promoter, BBa_E0840 - GFP generator and BBa_I13522 - pTet GFP]. I cultured the best colony in each plate in 5ml culture tubes with 1x ampicillin added and incubated at 37 &deg;C and 160 rpm at 5:30PM. So tomorrow we can proceed to the DNA extraction step.

Tim Vos
Since Sriram became busy with culturing the transformed colonies, I made 40x100 &micro;l aliquots into cool and sterile eppendorfs in laminar flow and stored in -80&deg;C freezer. In the freezer the competent cell containing eppendorfs were not labelled and kept in the box for each module. The 40 eppendorfs were separated as follows: 18 eppendorfs for 36 transformations in Delay device box, 11 eppendorfs for 22 transformations in Conjugation box and 11 eppendorfs for 22 transformations in SDP box.

Tim Weenink
Picked up my primers for the construction of the I-SceI cut site and phosphorylated the forward strand in a total volume of 20 µl. The amount of primer used was 1 µl of 10x dilution of the stock.

Then 1 µl of reverse primer was added and the primers were annealed by cooling the mixture from 95 to 20 degrees with a ramping temperature of 0.04 degrees per second