User:Wesley J. Houston/Notebook/MAP/2013/02/08

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(Autocreate 2013/02/08 Entry for User:Wesley_J._Houston/Notebook/MAP)
Current revision (18:55, 7 March 2013) (view source)
(February 8, 2013)
 
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{|{{table}} width="800"
{|{{table}} width="800"
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
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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: #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>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</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>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
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==Entry title==
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==February 8, 2013==
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* Insert content here...
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* Assembly: (5xGal4 : [Spacer : HSVtk TATA]) : (K : AmCyan : AmCyan : NLS : STOP : [polyA2]) (KAH201)/(KAH182); AmCyan reporter gene for chromatin proteins.
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----
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'''Assemblies'''
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* Note, KAH201 is in vector V0120, so add 3200 bp to the size.
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# KAH201/KAH182: (1) KAH201/(S/P)/203+3200 + (2) KAH182/(X/P)/1675
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> Digests (Fermentas FD)<br>
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{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
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|-valign="top"
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| <u>Reagent</u> || <u>Volume</u> || &nbsp;
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| 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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|-
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| DNA (plasmid) || 20 μL
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|-
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| 10x buffer || 3.0
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|-
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| enzyme 1 || 1.0
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|-
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| enzyme 2 || 1.0
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|-
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| dH<sub>2</sub>O || 5.0 μL
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|-
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| &nbsp; || 30 μL --> 37°C/ ~15 min.
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|}
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> Measure conc.'s of gel purified fragments
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{| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table -->
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|-
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| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
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|-
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| 1. KAH201 (S/P) || 0.048 || 1.88 || 48.1
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|-
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| 2. KAH182 (X/P) || 0.008 || 1.93 || 8.1
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|}
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> Ligations (corrected version, 3/04/13)
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{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table -->
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| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> 02/20/13</font>
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|-
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| 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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|-
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| 2. KAH201 (S/P)/3403, 25 ng || 0
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|}
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{| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->
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| &nbsp;            || 1    || 2    ||
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|-
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| Insert DNA (KAH182)        || 3.0  || ---  ||
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|-
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| Vector DNA (KAH201)        || 0.5  || 0.5  ||
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|-
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| 2x lgn buf (Roche) || 5.0  || 5.0  ||
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|-
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| T4 ligase (NEB)    || 1.0  || 1.0  ||
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|-
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| dH<sub>2</sub>O    ||  0.5 ||  3.5 ||
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|-
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| &nbsp;            || 10 μL || 10 μL ||
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|}
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* Follow quick transformation protocol
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* Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar
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* No colonies were formed, this will be repeated. See 2/26/13 entry.

Current revision

Building a Reporter Gene Main project page
Previous entry      Next entry

February 8, 2013

  • 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.
  1. KAH201/KAH182: (1) KAH201/(S/P)/203+3200 + (2) KAH182/(X/P)/1675

> Digests (Fermentas FD)


Reagent Volume   Results of the electrophoresis
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 20 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O 5.0 μL
  30 μL --> 37°C/ ~15 min.


> Measure conc.'s of gel purified fragments

Sample OD260 260/280 ng/μL
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)

Ligation Plate results (lig : neg crtl) 02/20/13
1. KAH201 (S/P)/3403, 25 ng + KAH182 (X/P)/1675, 26 ng 0
2. KAH201 (S/P)/3403, 25 ng 0
  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
dH2O 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.



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