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''' | ||
* BsmBI cuts the DNA fragments and creates complementary overhangs. | |||
* Complementary sticky ends anneal via base pairing. | |||
* T4 ligase seals gaps in the phosphodiester DNA backbone. | |||
{| {{table}} | |||
| bgcolor="grey" | Reagent | |||
| bgcolor="grey" | Vol. | |||
| rowspan=7 | '''Thermal cycling''' | |||
* [45°C, 2 min.; 16°C 5 min.] x25 | |||
* 60°C, 10 min. | |||
* 80°C, 20 min. | |||
* 4°C, ∞ | |||
|- | |||
| 20 fmol (1 μL) of each DNA part || up to 8.0 | |||
|- | |||
| 10x T4 ligase buffer (Promega) || 1.0 | |||
|- | |||
| T4 ligase (NEB) || 0.25 | |||
|- | |||
| BsmBI || 0.5 | |||
|- | |||
| dH<sub>2</sub>O || 0.25 | |||
|- | |||
| || 10.0 μL | |||
|} | |||
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table --> | |||
|- valign="top" | |||
| bgcolor="grey" | Reagent | |||
| bgcolor="grey" | 1 | |||
| bgcolor="grey" | 2 | |||
| bgcolor="grey" | 3 | |||
|- | |||
| gg2 pSB1A3 || 1.0 || 1.0 || 1.0 | |||
|- | |||
| gg3 hPCD || 1.0 || 1.0 || 1.0 | |||
|- | |||
| BL02 || 1.0 || --- || --- | |||
|- | |||
| BL03 || --- || 1.0 || --- | |||
|- | |||
| BL04 || --- || --- || 1.0 | |||
|- | |||
| 10x ligase buffer || 1.0 || 1.0 || 1.0 | |||
|- | |||
| NEB T4 lgase || 0.25 || 0.25 || 1.0 | |||
|- | |||
| BsmBI || 0.5 || 0.5 || 0.5 | |||
|- | |||
| dH<sub>2</sub>O || 5.25 || 5.25 || 5.25 | |||
|- | |||
| Total|| 10.0 || 10.0 | |||
|} | |||
<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.'' | |||
Revision as of 13:13, 9 February 2013
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2/8/2013
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