Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2013/03/11

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Entry title

came in around 10am to check plates. some grew, picked colonies and grew up in lb amp
miniprepped 8-1, 8-2, 8-3

colony PCR for ligation of Pconst - B0034 - C0061

10 colonies

picked, streaked on amp plate, swirled in 15ul water, heated at 95deg for 5 minutes, spun at 12g for 1 minute, added 'sup' to 15ul GoTAQ MM + biobrick primers

controls: 8-1 miniprep and Pconst+RFP miniprep

Growing the senders!

picked 3 colonies from each BL21 transformation plate and growing up in 2ml LB+amp

grew from 10am to 6 pm

sat on my bench until 7pm

added 1ml of culture to 4ml of media in round bottom tubes

shaking at 37

picked some colors without promoters from 2011 plate

ligations didn't have colonies yet

came in a 7PM

here's what grew from what was picked this morning:

1-2-1 no
1-2-2 no
2-2-1 no
2-2-2 yes
4-2-1 yes
4-2-2 yes
7-2-1 yes
7-2-2 no
10-2-1 yes
10-2-2 no
12-1-1 yes
12-1-2 no

some of the plates have more colonies. will run colony PCR to see what might be positive if these mini preps don't look good.
cut mini preps

2 X & P

yesterday's ligation:

Part colonies
Pconst bb control 3 colonies
Pconst + RBS + C0061 >20 colonies
Pconst + RBS + C0078 4-6 colonies (had low yield on gel extraction)
Pconst RFP bb control 3 colonies
Pconst RFP + K081023 2 colonies

picked more colonies from ligations and parts i pulled out yesterday

transformed some of the mini preps from today into BL21

2ul of mini prep into 10ul water

added 20ul BL21 cells

incubated on ice for 8-10 minutes

heat shocked at 42deg for 45sec

iced for 2 minutes


K082035 (2-2-2)

K084007 (4-2-1 and 4-2-2)

K145254 (7-2-1)