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

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(February 8, 2013)
(February 8, 2013)
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----
----
'''Assemblies'''
'''Assemblies'''
-
# KAH201/KAH182: (1) KAH201/(S/P)/203 + (2) KAH182/(X/P)/1675
+
# KAH201/KAH182: (1) KAH201/(X/P)/203 + (2) KAH182/(S/P)/1675
> Digests (Fermentas FD)<br>
> Digests (Fermentas FD)<br>
Line 22: Line 22:
| 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:KAH113012_gel1.jpg‎ |150px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
-
| DNA (plasmid) || up to 25 μL
+
| DNA (plasmid) || 15 to 25 μL
|-
|-
| 10x buffer || 3.0
| 10x buffer || 3.0
Line 30: Line 30:
| enzyme 2 || 1.0
| enzyme 2 || 1.0
|-
|-
-
| dH<sub>2</sub>O || ---
+
| dH<sub>2</sub>O || 0 to 10 μL
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~15 min.
| &nbsp; || 30 μL --> 37°C/ ~15 min.
Line 41: Line 41:
| <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 (X/P) || TBD || TBD || TBD
|-
|-
-
| 2. KAH182 (X/P) || TBD || TBD || TBD
+
| 2. KAH182 (S/P) || TBD || TBD || TBD
|}
|}
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| <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>
|-
|-
-
| 1. KAH201 (S/P)/203, 8 ng + KAH182 (X/P)/1675, 25 ng || <font color="blue">KAH3/MV2 >10:1 (Pick 2)</font>
+
| 1. KAH201 (X/P)/203, x ng + KAH182 (S/P)/1675, x ng || <font color="blue">---</font>
|-
|-
-
| 2. KAH182 (X/P)/ 25 ng || zero colonies
+
| 2. KAH182 (S/P)/ 25 ng || ---
|}
|}

Revision as of 01:47, 7 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/(X/P)/203 + (2) KAH182/(S/P)/1675

> Digests (Fermentas FD)


Reagent Volume   Hover name
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 15 to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O 0 to 10 μL
  30 μL --> 37°C/ ~15 min.


> Measure conc.'s of gel purified fragments

Sample OD260 260/280 ng/μL
1. KAH201 (X/P) TBD TBD TBD
2. KAH182 (S/P) TBD TBD TBD


> Ligations (edit after ligation is done)

Ligation Plate results (lig : neg crtl) 11/31/12
1. KAH201 (X/P)/203, x ng + KAH182 (S/P)/1675, x ng ---
2. KAH182 (S/P)/ 25 ng ---
  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



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