User:Wesley J. Houston/Notebook/MAP/2013/02/08: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Building a Reporter Gene</span> | ||
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | |style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | ||
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== | ==February 8, 2013== | ||
* Insert | * Assembly: (5xGal4 : [Spacer : HSVtk TATA]) : (K : AmCyan : AmCyan : NLS : STOP : [polyA2]) (KAH201)/(KAH182); AmCyan reporter gene for chromatin proteins. | ||
---- | |||
'''Assemblies''' | |||
* Note, KAH201 is in vector V0120, so add 3200 bp to the size. | |||
# KAH201/KAH182: (1) KAH201/(S/P)/203+3200 + (2) KAH182/(X/P)/1675 | |||
> Digests (Fermentas FD)<br> | |||
{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table --> | |||
|-valign="top" | |||
| <u>Reagent</u> || <u>Volume</u> || | |||
| rowspan="9" | [[Image:Electrophoresis1.jpg |150px|Results of the electrophoresis]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |||
| DNA (plasmid) || 20 μL | |||
|- | |||
| 10x buffer || 3.0 | |||
|- | |||
| enzyme 1 || 1.0 | |||
|- | |||
| enzyme 2 || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || 5.0 μL | |||
|- | |||
| || 30 μL --> 37°C/ ~15 min. | |||
|} | |||
> Measure conc.'s of gel purified fragments | |||
{| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table --> | |||
|- | |||
| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u> | |||
|- | |||
| 1. KAH201 (S/P) || 0.048 || 1.88 || 48.1 | |||
|- | |||
| 2. KAH182 (X/P) || 0.008 || 1.93 || 8.1 | |||
|} | |||
> Ligations (corrected version, 3/04/13) | |||
{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table --> | |||
| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> 02/20/13</font> | |||
|- | |||
| 1. KAH201 (S/P)/<font color="blue">3403</font>, 25 ng + KAH182 (X/P)/1675, 26 ng || <font color="blue">0</font> | |||
|- | |||
| 2. KAH201 (S/P)/3403, 25 ng || 0 | |||
|} | |||
{| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table --> | |||
| || 1 || 2 || | |||
|- | |||
| Insert DNA (KAH182) || 3.0 || --- || | |||
|- | |||
| Vector DNA (KAH201) || 0.5 || 0.5 || | |||
|- | |||
| 2x lgn buf (Roche) || 5.0 || 5.0 || | |||
|- | |||
| T4 ligase (NEB) || 1.0 || 1.0 || | |||
|- | |||
| dH<sub>2</sub>O || 0.5 || 3.5 || | |||
|- | |||
| || 10 μL || 10 μL || | |||
|} | |||
* Follow quick transformation protocol | |||
* Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar | |||
* No colonies were formed, this will be repeated. See 2/26/13 entry. | |||
Revision as of 15:55, 7 March 2013
Building a Reporter Gene | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
February 8, 2013
Assemblies
> Digests (Fermentas FD)
|