Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/12/05: Difference between revisions
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(Autocreate 2012/12/05 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes) |
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== | ==December 5, 2012== | ||
* | '''Results from last 11/23/12 ligation''' | ||
* None of the colonies on any plates grew | |||
* Will retry cloning for each (H2B, LOV) by using the Gibson assembly method | |||
---- | |||
* Repeat PCR of H2B and LOV inserts with new primers | |||
* Note: Primers add a XbaI site upstream and the SpeI site downstream | |||
* Final Gibson assembly products | |||
# pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end | |||
# pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end | |||
*For H2B-LOV construct | |||
# H2B-GFP plasmid + 1A3/H2B fwd + H2B/LOV rev | |||
# LOV plasmid + H2B/LOV fwd + LOV+his/1A3 rev | |||
*For LOV-H2B construct | |||
# H2B-GFP plasmid + 1A3/LOV fwd + LOV/H2B rev | |||
# LOV plasmid + LOV/H2B fwd + H2B+his/1A3 rev | |||
Performed table reactions twice: once for those used in H2B-LOV construct, once for those used in LOV-H2B construct | |||
{| class="wikitable" border=1 cellpadding="5" cellspacing="0" | |||
|- | |||
| Reagents || H2B || LOV | |||
|- | |||
| Plasmid DNA || 0.5 μL || 0.5 | |||
|- | |||
| primer 1 (10 μM) || 1.0 || 1.0 | |||
|- | |||
| primer 2 (10 μM) || 1.0 || 1.0 | |||
|- | |||
| 2x GoTaq mix || 12.5 || 12.5 | |||
|- | |||
| dH<sub>2</sub>O || 10.0 || 10.0 | |||
|- | |||
| Total || 25.0 || 25.0 | |||
|} | |||
PCR program: | |||
* 95°C, 3 min. | |||
* [95°C, 30 sec; 57°C, 30 sec.; 72°C, 30 sec.] x35 cycles | |||
* 72°C, 3 min. | |||
* 4°C ∞ | |||
* Clean up PCR products using the Zymo Clean and Concentrator kit | |||
* Be sure to elute using 15 μL dH<sub>2</sub>O | |||
{| class="wikitable" border=1 cellpadding="5" cellspacing="0" | |||
|- | |||
|Construct || Product || 260 || 280 || 260/280 || ng/μL | |||
|- | |||
|H2B-LOV || H2B 1 || 0.061 || 0.033 || 1.863 || 60.545 | |||
|- | |||
|H2B-LOV || H2B 2 || 0.056 || 0.03 || 1.857 || 56.22 | |||
|- | |||
|H2B-LOV || LOV 1 || 0.063 || 0.033 || 1.903 || 62.673 | |||
|- | |||
|H2B-LOV || LOV 2 || 0.06 || 0.04 || 1.85 || 59.844 | |||
|- | |||
|LOV-H2B || H2B 1 || 0.072 || 0.033 || 1.807 || 72.381 | |||
|- | |||
|LOV-H2B || H2B 2 || 0.06 || 0.036 || 1.799 || 59.925 | |||
|- | |||
|LOV-H2B || LOV 1 || 0.006 || 0.036 || 1.849 || 65.881 | |||
|- | |||
|LOV-H2B || LOV 2 || 0.065 || 0.035 || 1.839 || 65.285 | |||
|} | |||
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Revision as of 06:46, 8 December 2012
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December 5, 2012Results from last 11/23/12 ligation
Performed table reactions twice: once for those used in H2B-LOV construct, once for those used in LOV-H2B construct
PCR program:
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