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;"> Building a Reporter Gene</span> | |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"| | |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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---- | ---- | ||
'''Assemblies''' | '''Assemblies''' | ||
# KAH201/KAH182: (1) KAH201/(S/P)/203 + (2) KAH182/(X/P)/1675 | * 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> | > Digests (Fermentas FD)<br> | ||
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|-valign="top" | |-valign="top" | ||
| <u>Reagent</u> || <u>Volume</u> || | | <u>Reagent</u> || <u>Volume</u> || | ||
| rowspan="9" | [[Image: | | 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] | ||
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| DNA (plasmid) || | | DNA (plasmid) || 20 μL | ||
|- | |- | ||
| 10x buffer || 3.0 | | 10x buffer || 3.0 | ||
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| enzyme 2 || 1.0 | | enzyme 2 || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || 0 | | dH<sub>2</sub>O || 5.0 μL | ||
|- | |- | ||
| || 30 μL --> 37°C/ ~15 min. | | || 30 μL --> 37°C/ ~15 min. | ||
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| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u> | | <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u> | ||
|- | |- | ||
| 1. KAH201 (S/P) || | | 1. KAH201 (S/P) || 0.048 || 1.88 || 48.1 | ||
|- | |- | ||
| 2. KAH182 (X/P) || | | 2. KAH182 (X/P) || 0.008 || 1.93 || 8.1 | ||
|} | |} | ||
> Ligations ( | > Ligations (corrected version, 3/04/13) | ||
{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table --> | {| class="wikitable" border="0" cellspacing="3" <!-- Ligations table --> | ||
| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> | | <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> 02/20/13</font> | ||
|- | |- | ||
| 1. KAH201 (S/P)/ | | 1. KAH201 (S/P)/<font color="blue">3403</font>, 25 ng + KAH182 (X/P)/1675, 26 ng || <font color="blue">0</font> | ||
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| 2. | | 2. KAH201 (S/P)/3403, 25 ng || 0 | ||
|} | |} | ||
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| || 1 || 2 || | | || 1 || 2 || | ||
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| Insert DNA | | Insert DNA (KAH182) || 3.0 || --- || | ||
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| Vector DNA | | Vector DNA (KAH201) || 0.5 || 0.5 || | ||
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| 2x lgn buf (Roche) || 5.0 || 5.0 || | | 2x lgn buf (Roche) || 5.0 || 5.0 || | ||
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| T4 ligase (NEB) || 1.0 || 1.0 || | | T4 ligase (NEB) || 1.0 || 1.0 || | ||
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| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 0.5 || 3.5 || | ||
|- | |- | ||
| || 10 μL || 10 μL || | | || 10 μL || 10 μL || | ||
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* Follow quick transformation protocol | * Follow quick transformation protocol | ||
* Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar | * 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. | |||
Latest revision as of 22:25, 26 September 2017
Building a Reporter Gene | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
February 8, 2013
Assemblies
> Digests (Fermentas FD)
|