Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/02/08: Difference between revisions
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(Autocreate 2013/02/08 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes) |
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== | ==February 8, 2013== | ||
* | '''Type IIS Assembly | ||
* Attempt new assembly method because piecewise assembly was unsuccessful | |||
* Final assembly product: pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end | |||
* Primer design | |||
H2B | |||
1A3/H2B fwd 5'-cacaccaCGTCTCa TAGA ATGCCAGAGCCAGCG | |||
H2B/LOV rev 5'-cacaccaCGTCTCa CCAA CTTAGCGCTGGTGTA | |||
LOV | |||
LOV fwd 5'-cacaccaCGTCTCa TTGGCTACTACACTT | |||
LOV+his/1A3 rev 5'-cacaccaCGTCTCa TAGT ttagtggtgatggtgatgatgAAGTTCTTTTGCCGC | |||
* First, resuspend dry primer oligos (in blue-capped tubes) to 100 μM | |||
* Then make working solutions of 10 μM | |||
* Reactions: | |||
# H2B-GFP plasmid + 1A3/H2B fwd + H2B/LOV rev | |||
# LOV plasmid + LOV fwd + LOV+his/1A3 rev | |||
{| class="wikitable" border=1 cellpadding="5" cellspacing="0" | |||
|- | |||
| Reagents || H2B || LOV | |||
|- | |||
| Plasmid DNA || 0.2 μL || 0.2 | |||
|- | |||
| primer 1 (10 μM) || 1.0 || 1.0 | |||
|- | |||
| primer 2 (10 μM) || 1.0 || 1.0 | |||
|- | |||
| 2x GoTaq mix || 25.0 || 25.0 | |||
|- | |||
| dH<sub>2</sub>O || 22.8 || 22.8 | |||
|- | |||
| Total || 50.0 || 50.0 | |||
|} | |||
PCR program: | |||
* 95°C, 3 min. | |||
* [95°C, 30 sec; 57°C, 30 sec.; 72°C, 1 min.] x35 cycles | |||
* 72°C, 3 min. | |||
* 4°C ∞ | |||
{| class="wikitable" border=0 | |||
|- | |||
| <u>Expected:</u><br>1. H2B = 410 <br>2. LOV = 461 | |||
| <br> | |||
|} | |||
<center> | |||
[[Image:Gel_2-8-13.png|Results of the digests are shown.]] | |||
[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]] | |||
</center> | |||
<center> | |||
Results of the PCR product digests are shown, with H2B on the left and LOV on the right. The gel was loaded with 5 μL samples. | |||
</center> | |||
* The PCR products were purified with the Zymo DNA Clean & Concentrator Kit and eluted with 20 μL dH<sub>2</sub>O | |||
{| class="wikitable" border=1 cellpadding="5" cellspacing="0" | |||
|- | |||
| PCR Product || 260|| 280 || 260/280 || ng/μL | |||
|- | |||
| H2B || fill || fill || fill || fill | |||
|- | |||
| LOV || fill || fill || fill || fill | |||
|} | |||
Revision as of 05:39, 8 February 2013
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February 8, 2013Type IIS Assembly
H2B 1A3/H2B fwd 5'-cacaccaCGTCTCa TAGA ATGCCAGAGCCAGCG H2B/LOV rev 5'-cacaccaCGTCTCa CCAA CTTAGCGCTGGTGTA LOV LOV fwd 5'-cacaccaCGTCTCa TTGGCTACTACACTT LOV+his/1A3 rev 5'-cacaccaCGTCTCa TAGT ttagtggtgatggtgatgatgAAGTTCTTTTGCCGC
PCR program:
Results of the PCR product digests are shown, with H2B on the left and LOV on the right. The gel was loaded with 5 μL samples.
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