Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/02/08: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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== | ==February 8, 2013== | ||
* | '''Type IIS Assembly - Making PCR Products ''' | ||
* 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:VN_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. It was later found that the transparency used was blocking the UV light, so no bands could be seen. | |||
</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 || 0.078|| 0.043|| 1.81 || 77.995 | |||
|- | |||
| LOV || 0.08 || 0.044 || 1.817 || 80.443 | |||
|- | |||
| pSB1A3 || 0.023 || 0.014 || 1.727 || 23.386 | |||
|- | |||
|} | |||
Latest revision as of 22:25, 26 September 2017
Project name | Main project page Previous entry Next entry | |||||||||||||||||||||||||||||||||||||||||||
February 8, 2013Type IIS Assembly - Making PCR Products
1A3/H2B fwd 5'-cacaccaCGTCTCa TAGA ATGCCAGAGCCAGCG H2B/LOV rev 5'-cacaccaCGTCTCa CCAA CTTAGCGCTGGTGTA
LOV fwd 5'-cacaccaCGTCTCa TTGGCTACTACACTT LOV+his/1A3 rev 5'-cacaccaCGTCTCa TAGT ttagtggtgatggtgatgatgAAGTTCTTTTGCCGC
PCR program:
Results of the PCR product digests are shown. It was later found that the transparency used was blocking the UV light, so no bands could be seen.
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