IGEM:MIT/2006/Notebook/2006-11-1

Yeast Stuff

 * 1) Yeast strain (ACLY379) finally grew up overnight. Diluted back at 9:45 AM, and will commence transformation after SB lunch.
 * 2) I let the gel of yesterday's digest results run a bit longer to see the exact length of the vectors, and it appears that most of our vectors are just the backbone vector, no insert.
 * 3) But there are faint bands that might be the correct length. I'll rerun and gel extract, if the bands are visible enough.
 * 4) Transformation
 * 5) Use high-efficiency protocol because we want to give this transformation the best shot possible.
 * 6) Transform the amalagam of G5, G6, G7 digests from Oct. 31.

Assemblies

 * Have decent number of colonies for all 4 assemblies.
 * Barry is screening J45320.J45180. All others are either wrong (due to pSB1AT3-J45400 being wrong) or pointless at this time.

Overnights

 * Grew pSB1AT3-J45200 in the wrong media ... Amp-Cm.
 * pSB1AC3-J45250/pSB3K3-J45400 didn't grow (either one).
 * But the one started on 30th evening is grown up.
 * Neither the pSB1AK3-J45180 big or small colonies grew up in culture (reshma)
 * One of my 45180 colonies (big) did grow, the other (small) didn't (austin)

Streak tests

 * pSB1AT3-J45320 grows on Amp-Tet.
 * pSB3K3-J45400 grows on Kan.
 * pSB1AK3-J45180 grows on Kan.
 * pSB1AT3-J45120 grows on Amp-Cm and Amp-Tet.
 * pSB1AT3-J45400 grows on Amp-Cm but not Amp-Tet.
 * Agrees with my liquid culture results (austin)
 * Sent out "1AT3-J45400" for sequencing with VF2/VR (austin)
 * Jason clarified. This construct is totally wrong.  Reverting back to pSB3K3 version.
 * pSB1AC3-J45700 grows on Amp-Cm.
 * pSB1AC3-J45250 grows on Amp-Cm.
 * I got one colony of pSB1AT3-J45200 on an Amp plate. Maybe slight growth on Tet.

To do

 * Make electrically competent indole deficient strain (done)
 * Determine the best way to screen colonies. Find primers that can be used to determine if parts are in properly.
 * For construct J45320.J45180, use pchA mut-f and osmY-r
 * For construct J45250.J45400, use ATF1 mut-f and BAT2 mut-r
 * Grow up in liquid culture some colonies from each plate during day. (Barry)
 * Miniprep + transform indole(-) strain tonight. (cancelled)
 * Transform J45250 into IK cells. (Austin and Reshma).
 * Ligate and transform J45250.J45400 (from pSB3K3) (Austin and Reshma).
 * Grow cultures of (done, Reshma)
 * pSB3K3-J45400
 * pSB1AK3-J45180
 * pSB1AT3-J45200
 * pSB1AC3-J45250
 * pSB1AC3-J45700