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

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|-
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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"|<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"|[[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]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#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> || &nbsp;
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
| rowspan="9" | [[Image:KAH113012_gel1.jpg‎ |150px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
| 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) || 20 μL
| DNA (plasmid) || 20 μL
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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) || TBD || TBD || TBD
| 1. KAH201 (S/P) || 0.048 || 1.88 || 48.1
|-
|-
| 2. KAH182 (X/P) || TBD || TBD || TBD
| 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> mm/dd/yy</font>
| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> 02/20/13</font>
|-
|-
| 1. KAH201 (S/P)/203, 6 ng + KAH182 (X/P)/1675, 25 ng || <font color="blue">TBD</font>
| 1. KAH201 (S/P)/<font color="blue">3403</font>, 25 ng + KAH182 (X/P)/1675, 26 ng || <font color="blue">0</font>
|-
|-
| 2. KAH182 (X/P)/1675 25 ng || TBD
| 2. KAH201 (S/P)/3403, 25 ng || 0
|}
|}


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| &nbsp;            || 1    || 2    ||
| &nbsp;            || 1    || 2    ||
|-
|-
| Insert DNA         || TBD || ---  ||
| Insert DNA (KAH182)        || 3.0 || ---  ||
|-
|-
| Vector DNA         || TBD || TBD ||
| Vector DNA (KAH201)        || 0.5 || 0.5 ||
|-
|-
| 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  ||
|-
|-
| dH<sub>2</sub>O    || TBD || TBD ||
| dH<sub>2</sub>O    ||  0.5 ||  3.5 ||
|-
|-
| &nbsp;            || 10 μL || 10 μL ||
| &nbsp;            || 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

  • 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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