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

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> Ligations (edit after ligation is done)
> 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> mm/dd/yy</font>
|-
|-
| 1. KAH201 (X/P)/203, x ng + KAH182 (S/P)/1675, x ng || <font color="blue">---</font>
| 1. KAH201 (X/P)/203, x ng + KAH182 (S/P)/1675, x ng || <font color="blue">---</font>

Revision as of 23:01, 6 February 2013

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/(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) mm/dd/yy
1. KAH201 (X/P)/203, x ng + KAH182 (S/P)/1675, x ng ---
2. KAH182 (S/P)/ x 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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