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

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(February 8, 2013)
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> Ligations
+
> Ligations (edit after ligation is done)
 
{| 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> 11/31/12</font>
 
| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> 11/31/12</font>
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| &nbsp;            || 1    || 2    ||
 
| &nbsp;            || 1    || 2    ||
 
|-
 
|-
| Insert DNA        || 0.3 || ---  ||
+
| Insert DNA        || --- || ---  ||
 
|-
 
|-
| Vector DNA        || 0.3 || 0.3 ||
+
| Vector DNA        || --- || --- ||
 
|-
 
|-
| 2x lgn buf (Roche) || 5.0 || 5.0 ||
+
| 2x lgn buf (Roche) || --- || --- ||
 
|-
 
|-
| T4 ligase (NEB)    || 1.0 || 1.0 ||
+
| T4 ligase (NEB)    || --- || --- ||
 
|-
 
|-
| dH<sub>2</sub>O    || 3.4 || 3.7 ||
+
| dH<sub>2</sub>O    || --- || --- ||
 
|-
 
|-
 
| &nbsp;            || 10 μL || 10 μL ||
 
| &nbsp;            || 10 μL || 10 μL ||

Revision as of 20:51, 6 February 2013

Owwnotebook icon.png Building a Reporter Gene <html><img src="/images/9/94/Report.png" border="0" /></html> 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   Hover name
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  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 (edit after ligation is done)

Ligation Plate results (lig : neg crtl) 11/31/12
1. KAH201 (S/P)/203, 8 ng + KAH182 (X/P)/1675, 25 ng KAH3/MV2 >10:1 (Pick 2)
2. KAH182 (X/P)/ 25 ng zero colonies
  1 2
Insert DNA --- ---
Vector DNA --- ---
2x lgn buf (Roche) --- ---
T4 ligase (NEB) --- ---
dH2O --- ---
  10 μL 10 μL
  • Follow quick transformation protocol
  • Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar