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==
-
* 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.
----
----
'''Assemblies'''
'''Assemblies'''
-
# KAH201/KAH182: (1) KAH201/(X/P)/203 + (2) KAH182/(S/P)/1675
+
* Note, KAH201 is in vector V0120, so add 3200 bp to the size.
 +
# KAH201/KAH182: (1) KAH201/(S/P)/203+3200 + (2) KAH182/(X/P)/1675
> Digests (Fermentas FD)<br>
> Digests (Fermentas FD)<br>
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|-valign="top"
|-valign="top"
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
-
| 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:Electrophoresis1.jpg‎ |150px|Results of the electrophoresis]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
-
| DNA (plasmid) || 15 to 25 μL
+
| 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 || 0 to 10 μL
+
| dH<sub>2</sub>O || 5.0 μL
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~15 min.
| &nbsp; || 30 μL --> 37°C/ ~15 min.
Line 41: Line 42:
| <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 (X/P) || TBD || TBD || TBD
+
| 1. KAH201 (S/P) || 0.048 || 1.88 || 48.1
|-
|-
-
| 2. KAH182 (S/P) || TBD || TBD || TBD
+
| 2. KAH182 (X/P) || 0.008 || 1.93 || 8.1
|}
|}
-
> Ligations (edit after ligation is done)
+
> Ligations (corrected version, 3/04/13)
{| 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> 02/20/13</font>
|-
|-
-
| 1. KAH201 (X/P)/203, x ng + KAH182 (S/P)/1675, x ng || <font color="blue">---</font>
+
| 1. KAH201 (S/P)/<font color="blue">3403</font>, 25 ng + KAH182 (X/P)/1675, 26 ng || <font color="blue">0</font>
|-
|-
-
| 2. KAH182 (S/P)/ 25 ng || ---
+
| 2. KAH201 (S/P)/3403, 25 ng || 0
|}
|}
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| &nbsp;            || 1    || 2    ||
| &nbsp;            || 1    || 2    ||
|-
|-
-
| Insert DNA         || --- || ---  ||
+
| Insert DNA (KAH182)        || 3.0 || ---  ||
|-
|-
-
| Vector DNA         || --- || --- ||
+
| Vector DNA (KAH201)        || 0.5 || 0.5 ||
|-
|-
-
| 2x lgn buf (Roche) || --- || --- ||
+
| 2x lgn buf (Roche) || 5.0 || 5.0 ||
|-
|-
-
| T4 ligase (NEB)    || --- || --- ||
+
| T4 ligase (NEB)    || 1.0 || 1.0 ||
|-
|-
-
| dH<sub>2</sub>O    || --- || --- ||
+
| dH<sub>2</sub>O    ||  0.5 ||  3.5 ||
|-
|-
| &nbsp;            || 10 μL || 10 μL ||
| &nbsp;            || 10 μL || 10 μL ||
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* Follow quick transformation protocol
* Follow quick transformation protocol
* Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar
* 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.

Current revision

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

  • 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   Results of the electrophoresis
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 (S/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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