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

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(February 8, 2013)
(February 8, 2013)
Line 60: Line 60:
|              || 1    || 2    ||
|              || 1    || 2    ||
|-
|-
-
| Insert DNA (KAH182)        || 0.74 || ---  ||
+
| Insert DNA (KAH182)        || 3.0 || ---  ||
|-
|-
-
| Vector DNA (KAH201)        || 0.52 || 0.52 ||
+
| 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    || 2.74 ||  3.48 ||
+
| dH<sub>2</sub>O    || 0.5 ||  3.5 ||
|-
|-
| &nbsp;            || 10 μL || 10 μL ||
| &nbsp;            || 10 μL || 10 μL ||

Revision as of 13:58, 4 March 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

  • 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   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 (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 (X/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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