Haynes Lab:Notebook/Engineering PC-TFs/2013/02/08: Difference between revisions
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== | ==2/8/2013== | ||
* * '''Perform BsmBI/ T4 ligase mediated assembly''' | * * '''Perform BsmBI/ T4 ligase mediated assembly''' | ||
* BsmBI cuts the DNA fragments and creates complementary overhangs. | * BsmBI cuts the DNA fragments and creates complementary overhangs. | ||
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<font size=3>'''Bacterial transformation'''</font> | |||
* Added total volume (10.0 μL) to 50 μL chemically competent cells (e.g., BL21) in a 2.0 mL tube. | |||
* Incubated on ice for 2 min., heat shock at 42°C for exactly 45 sec., and placed on ice. | |||
* Added 800 μL sterile SOC medium. | |||
* Grow with shaking at 37°C for 30 min.(''Taped tubes to the shaker rack.'') | |||
* Pelleted the cells at top speed in a microcentrifuge for 3 min. at room temperature. | |||
* Discarded the supernatant. Resuspended the cells in 100 μL LB + antibiotic. | |||
* Plate cells on pre-warmed LB agar + antibiotic. (''I plated 100 μL of the cells. Then put beads on plates and shook.) Protocol didn't specify.'') Grow overnight at 37°C. | |||
* Check results tomorrow. | |||
[[Image:plates_1_8_2013.jpg|400px||left|]] | |||
*''Still nothing. I'm going to start everything over and see if I can troubleshoot. Vi should be working on Type IIS Assembly this week. So I should have something going by the end of next week.'' | |||
Latest revision as of 22:26, 26 September 2017
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2/8/2013
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