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

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----
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'''Assemblies'''
'''Assemblies'''
# KAH201/KAH182: (1) KAH201/(X/P)/203 + (2) KAH182/(S/P)/1675
# KAH201/KAH182: (1) KAH201/(E/S)/203 + (2) KAH182/(E/X)/1675


> Digests (Fermentas FD)<br>
> Digests (Fermentas FD)<br>
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 (X/P) || TBD || TBD || TBD
| 1. KAH201 (E/S) || TBD || TBD || TBD
|-
|-
| 2. KAH182 (S/P) || TBD || TBD || TBD
| 2. KAH182 (E/X) || TBD || TBD || TBD
|}
|}


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| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</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 (E/S)/203, x ng + KAH182 (E/X)/1675, x ng || <font color="blue">---</font>
|-
|-
| 2. KAH182 (S/P)/ x ng || ---
| 2. KAH182 (E/X)/ x ng || ---
|}
|}


Revision as of 23:11, 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/(E/S)/203 + (2) KAH182/(E/X)/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 (E/S) TBD TBD TBD
2. KAH182 (E/X) TBD TBD TBD


> Ligations (edit after ligation is done)

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. KAH201 (E/S)/203, x ng + KAH182 (E/X)/1675, x ng ---
2. KAH182 (E/X)/ 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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