Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/06: 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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| colspan="2"| | | colspan="2"| | ||
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{|border="1" cellpadding="5" cellspacing="0" align="center" | {|border="1" cellpadding="5" cellspacing="0" align="center" | ||
|+Restriction Digest Reagents | |||
|- | |- | ||
! scope="col" style="background:#efefef;" | Reagent | ! scope="col" style="background:#efefef;" | Reagent | ||
! scope="col" style="background:#efefef;" | H2B-GFP/EcoRI | ! scope="col" style="background:#efefef;" | H2B-GFP/EcoRI | ||
! scope="col" style="background:#efefef;" | H2B-GFP/PstI | ! scope="col" style="background:#efefef;" | H2B-GFP/PstI | ||
! scope="col" style="background:#efefef;" | LOV/ | ! scope="col" style="background:#efefef;" | LOV/PstI | ||
|- | |- | ||
|DNA(plasmid) | |DNA(plasmid) | ||
|2.0 | |2.0 | ||
|2.0 | |2.0 | ||
|2.0 | |2.0 | ||
|- | |- | ||
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|- | |- | ||
|EcoRI | |EcoRI | ||
|1 | |1.0 | ||
|0 | |||
|0 | |0 | ||
|- | |- | ||
|PstI | |PstI | ||
|0 | |0 | ||
|1 | |1.0 | ||
|0 | |1.0 | ||
|- | |||
|dH<sub>2</sub>O | |dH<sub>2</sub>O | ||
|10.5 | |10.5 | ||
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|- | |- | ||
|Total | |Total | ||
|15 | |15.0 | ||
|15 | |15.0 | ||
|15 | |15.0 | ||
|} | |} | ||
[[Image: | |||
{| class="wikitable" border=0 | |||
|- | |||
| <u>Expected:</u><br>1. H2B-GFP/ EcoRI = 4885, 210 <br>2. H2B-GFP/ PstI = 5101<br>3. LOV/PstI = 251 | |||
| <br> | |||
|} | |||
<center> | |||
[[Image:VN_Gel_11-6-12.png|150px|Results of the digests are shown.]] | |||
[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]] | |||
</center> | |||
<center> | |||
Results of the digests are shown. | |||
</center> | |||
---- | |||
*'''[[User:Karmella Haynes|---Karmella]] 16:01, 7 November 2012 (EST)''': Note: Please re-do LOV/PstI | |||
<br> | |||
*'''[[User:Karmella Haynes|---Karmella]] 16:01, 7 November 2012 (EST)''': Here is a reaction table for PCR | |||
* First, resuspend dry primer oligos (in blue-capped tubes) to 100 μM | |||
* Then make working solutions of 10 μM | |||
* Reactions: | |||
# H2B-GFP plasmid + BB_H2B fwd + BB_H2B rev | |||
# LOV plasmid + BB_LOV fwd + BB_LOV rev | |||
{| 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 || 10.5 || 10.5 | |||
|} | |||
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 ∞ | |||
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Latest revision as of 22:13, 26 September 2017
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November 6, 2012
Results of the digests are shown.
PCR program:
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