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

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Autocreate 2013/02/08 Entry for User:Wesley_J._Houston/Notebook/MAP)
(→February 8, 2013)
(18 intermediate revisions not shown.)
Line 1: Line 1:
{|{{table}} width="800"
{|{{table}} width="800"
|-
|-
-
|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;"> 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>}}
|-
|-
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
-
==Entry title==
+
==February 8, 2013==
-
* Insert content here...
+
* Assembly: (5xGal4 : [Spacer : HSVtk TATA]) : (K : AmCyan : AmCyan : NLS : STOP : [polyA2]) (KAH201)/(KAH182); AmCyan reporter gene for chromatin proteins.
 +
 
 +
 
 +
----
 +
'''Assemblies'''
 +
# KAH201/KAH182: (1) KAH201/(S/P)/203 + (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> || &nbsp;
 +
| rowspan="9" | [[Image:KAH113012_gel1.jpg‎ |150px|Placeholder Image]]<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
 +
|-
 +
| &nbsp; || 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
 +
{| 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>
 +
|-
 +
| 1. KAH201 (S/P)/203, 25 ng + KAH182 (X/P)/1675, 6 ng || <font color="blue">TBD</font>
 +
|-
 +
| 2. KAH182 (X/P)/1675 6 ng || TBD
 +
|}
 +
 
 +
{| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->
 +
| &nbsp;            || 1    || 2    ||
 +
|-
 +
| Insert DNA        || 0.74  || ---  ||
 +
|-
 +
| Vector DNA        || 0.52  || 0.52  ||
 +
|-
 +
| 2x lgn buf (Roche) || 5.0  || 5.0  ||
 +
|-
 +
| T4 ligase (NEB)    || 1.0  || 1.0  ||
 +
|-
 +
| dH<sub>2</sub>O    || 2.74 ||  3.48 ||
 +
|-
 +
| &nbsp;            || 10 μL || 10 μL ||
 +
|}
 +
 
 +
* Follow quick transformation protocol
 +
* Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar
 +
 

Revision as of 16:59, 21 February 2013

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

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

> Digests (Fermentas FD)


Reagent Volume   Placeholder Image
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

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. KAH201 (S/P)/203, 25 ng + KAH182 (X/P)/1675, 6 ng TBD
2. KAH182 (X/P)/1675 6 ng TBD
  1 2
Insert DNA 0.74 ---
Vector DNA 0.52 0.52
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 2.74 3.48
  10 μL 10 μL
  • Follow quick transformation protocol
  • Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar



Retrieved from "http://www.openwetware.org/wiki/User:Wesley_J._Houston/Notebook/MAP/2013/02/08"
Personal tools