Lablog

Before 11.4. "in silico EXPERIMENTS"
Brainstorming.
Discussing what plasmids, primers, enzymes, and chemicals we need.

25 weeks to go
11.4. "start the WET LAB work"
We obtained TAL-A, TAL-B, TAL-C, TAL-D, and Nic-TAL from the AddGene and from Registry and KRAB and VP16 from the lab sources.
Plasmids were transformed into DH5a and the bacteria were plated on ampicilin plates.
Single colonies were inoculated into minipreps.
We isolated the plasmids in larger quantities.
Competent cells (DHA5α, DB3.1) were prepared.
Preparation of LB media.
Cloning of TAL regulators (TAL+VP16).
Primers ordering.
Cloning repressor reporters 7xNicTAL+7xTAL95 BS-CMV-mCit.

24 weeks (6 months - half a year) to go
16.4. - 22.4. "TAL week"
TAL-A, TAL-B, TAL-C, TAL-D, Nic-TAL and KRAB fragments were amplified using PCR.
We tried to ligate KRAB upstream, downsteram and on both positions of all TAL constructs.
PCR ligation was used, but failed repeatedly.
:-( Performing multiple PCR-ligations trying to join TAL molecule and VP16 activation domain.
Cloning repressor reporters 7xNicTAL+7xTAL95 BS-CMV-mCit.

23 to go
23.4. - 29.4. "another TAL week"
Cloning of TAL regulators.
Unsuccessful cloning of activating reporters 7xNicTAL+7xTAL95 BS-pMin-mCit.

22 to go
30.4. - 6.5. "and another TAL week"
Performing multiple colony-PCRs.
Repeatedly trying to get less back ligations.
Another week of unsuccessful cloning of activating reporters 7xNicTAL+7xTAL95 BS-pMin-mCit.

21 to go
7.5. - 13.5. "light at the end of tunnel"
First samples of TAL regulators were send for sequencing.
:-)

20 weeks (5 months) to go
14.5. - 20.5. "TAL success-I"
First TAL activators were cloned and their sequence confirmed.
Second round of TAL regulators send for sequencing.
Went straight to performing PCR for cloning mCit into pGL vector. A note from one of our advisors