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

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<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
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==February 8, 2013==
==February 8, 2013==
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* Assembly: 5xGal4 : [Spacer : HSVtk TATA] : K : AmCyan : AmCyan : NLS : STOP : [polyA2] KAH201/KAH182; AmCyan reporter gene for chromatin proteins.
+
* Assembly: (5xGal4 : [Spacer : HSVtk TATA]) : (K : AmCyan : AmCyan : NLS : STOP : [polyA2]) (KAH201)/(KAH182); AmCyan reporter gene for chromatin proteins.
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|-valign="top"
|-valign="top"
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
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| 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]
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| 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]
|-
|-
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| DNA (plasmid) || up to 25 μL
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| 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 || ---
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| dH<sub>2</sub>O || 5.0 μL
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~15 min.
| &nbsp; || 30 μL --> 37°C/ ~15 min.
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> Ligations (edit after ligation is done)
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> Ligations
{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table -->
{| 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> 11/31/12</font>
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| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
|-
|-
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| 1. KAH201 (S/P)/203, 8 ng + KAH182 (X/P)/1675, 25 ng || <font color="blue">KAH3/MV2 >10:1 (Pick 2)</font>
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| 1. KAH201 (S/P)/203, 6 ng + KAH182 (X/P)/1675, 25 ng || <font color="blue">TBD</font>
|-
|-
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| 2. KAH182 (X/P)/ 25 ng || zero colonies
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| 2. KAH182 (X/P)/1675 25 ng || TBD
|}
|}
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| &nbsp;            || 1    || 2    ||
| &nbsp;            || 1    || 2    ||
|-
|-
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| Insert DNA        || --- || ---  ||
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| Insert DNA        || TBD || ---  ||
|-
|-
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| Vector DNA        || --- || --- ||
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| Vector DNA        || TBD || TBD ||
|-
|-
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| 2x lgn buf (Roche) || --- || --- ||
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| 2x lgn buf (Roche) || 5.0 || 5.0 ||
|-
|-
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| T4 ligase (NEB)    || --- || --- ||
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| T4 ligase (NEB)    || 1.0 || 1.0 ||
|-
|-
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| dH<sub>2</sub>O    || --- || --- ||
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| dH<sub>2</sub>O    || TBD || TBD ||
|-
|-
| &nbsp;            || 10 μL || 10 μL ||
| &nbsp;            || 10 μL || 10 μL ||

Revision as of 18:41, 15 February 2013

Building a Reporter Gene Main project page
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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) TBD TBD TBD
2. KAH182 (X/P) TBD TBD TBD


> Ligations

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



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