Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2013/03/11: Difference between revisions
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==Entry title== | ==Entry title== | ||
came in around 10am to check plates. some grew, picked colonies and grew up in lb amp | |||
<br> | |||
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 | |||
<br> | |||
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 | |||
<br> | |||
picked some colors without promoters from 2011 plate | |||
<br> | |||
ligations didn't have colonies yet | |||
came in a 7PM | |||
here's what grew from what was picked this morning: | |||
{| {{Table}} | |||
|- | |||
|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. | |||
<br> | |||
cut mini preps | |||
2 X & P | |||
yesterday's ligation: | |||
{| {{table}} | |||
|- | |||
|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 | |||
plated | |||
K082035 (2-2-2) | |||
K084007 (4-2-1 and 4-2-2) | |||
K145254 (7-2-1) | |||
Revision as of 16:17, 28 May 2013
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Entry titlecame in around 10am to check plates. some grew, picked colonies and grew up in lb amp
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
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:
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.
2 X & P
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 plated K082035 (2-2-2) K084007 (4-2-1 and 4-2-2) K145254 (7-2-1)
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