User:Wesley J. Houston/Notebook/MAP/2013/03/04: Difference between revisions

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==3/4/13==
==3/4/13==
*Second ligation was successful.
*Second ligation was successful.
* 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.
# KAH201/KAH182: (1) KAH201/(S/P)/203+3200 + (2) KAH182/(X/P)/1675
> Digests (Fermentas FD)<br>
{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
|-valign="top"
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
| 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) || 20 μL
|-
| 10x buffer || 3.0
|-
| enzyme 1 || 1.0
|-
| enzyme 2 || 1.0
|-
| dH<sub>2</sub>O || 5.0 μL
|-
| &nbsp; || 30 μL --> 37°C/ ~15 min.
|}
> Measure conc.'s of gel purified fragments
{| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table -->
|-
| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
|-
| 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)
> Ligations (corrected version, 3/04/13)

Revision as of 15:56, 7 March 2013

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3/4/13

  • Second ligation was successful.
  • 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) 03/04/13
1. KAH201 (S/P)/3403, 25 ng + KAH182 (X/P)/1675, 26 ng 10
2. KAH201 (S/P)/3403, 25 ng 0



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