Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/02/11: Difference between revisions
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== | ==February 9, 2013== | ||
* | '''Golden Gate Assembly''' | ||
* Mediated assembly | |||
** The volume of purified DNA (x) needed to dilute in a final volume of 20 μL = length in bp ÷ measured ng/μL * 20 fmols/μL * 650 fg/fmol ÷ 1,000,000 fg/ng * 20 μL final volume | |||
** Formula: x = length in bp ÷ measured ng/μL * 0.013 * 20 | |||
{| class="wikitable" border=1 cellpadding="5" cellspacing="0" | |||
|- | |||
| DNA || length (bp) || ng/μL || vol. || dH<sub>2</sub>O | |||
|- | |||
| H2B || 410 || 77.995 || 1.4 || 18.6 | |||
|- | |||
| LOV || 461 || 80.443 || 1.5 || 18.5 | |||
|- | |||
| pSB1A3 || 2000 || 77.4 || 6.7 || 13.3 | |||
|} | |||
{| class="wikitable" border=1 cellpadding="5" cellspacing="0" | |||
|- | |||
| Reagent || Assembly || Control | |||
|- | |||
| 20 fmol H2B || 1.0 || 0 | |||
|- | |||
| 20 fmol LOV || 1.0 || 0 | |||
|- | |||
| 20 fmol pSB1A3 || 1.0 || 1.0 | |||
|- | |||
| 10x T4 ligase buffer (Promega) || 1.0 || 1.0 | |||
|- | |||
| T4 ligase (NEB) || 0.25 || 0.25 | |||
|- | |||
| BsmBI || 0.5 || 0.5 | |||
|- | |||
| dH<sub>2</sub>O || 5.25 || 7.25 | |||
|- | |||
| Total vol. || 10.0 || 10.0 | |||
|} | |||
* Bacterial transformation | |||
** Add total volume (10.0 μL) to 50 μL chemically competent cells (e.g., BL21) in a 2.0 mL tube. | |||
** Incubate on ice for 2 min., heat shock at 42°C for exactly 45 sec., immediately place on ice. | |||
** Add 800 μL sterile SOC medium. | |||
** Grow with shaking at 37°C for 30 min. | |||
** Pellet the cells at top speed in a microcentrifuge for 3 min. at room temp. | |||
** Discard the supernatant. Resuspend the cells in 100 μL LB + antibiotic. | |||
** Plate cells on pre-warmed LB agar + antibiotic. Grow overnight at 37°C. Quick-transormation (e.g., DH5α-Turbo) is not recommended | |||
Revision as of 06:41, 7 May 2013
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February 9, 2013Golden Gate Assembly
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