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

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(→February 8, 2013)
(→February 8, 2013)
Line 51: Line 51:
| <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 (S/P)/203, 6 ng + KAH182 (X/P)/1675, 25 ng || <font color="blue">TBD</font>
+
| 1. KAH201 (S/P)/203, 25 ng + KAH182 (X/P)/1675, 6 ng || <font color="blue">TBD</font>
|-
|-
-
| 2. KAH182 (X/P)/1675 25 ng || TBD
+
| 2. KAH182 (X/P)/1675 6 ng || TBD
|}
|}

Revision as of 16:59, 21 February 2013

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

  1. KAH201/KAH182: (1) KAH201/(S/P)/203 + (2) KAH182/(X/P)/1675

> Digests (Fermentas FD)


Reagent Volume   Placeholder Image
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

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. KAH201 (S/P)/203, 25 ng + KAH182 (X/P)/1675, 6 ng TBD
2. KAH182 (X/P)/1675 6 ng TBD
  1 2
Insert DNA 0.74 ---
Vector DNA 0.52 0.52
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 2.74 3.48
  10 μL 10 μL
  • Follow quick transformation protocol
  • Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar



Retrieved from "http://www.openwetware.org/wiki/User:Wesley_J._Houston/Notebook/MAP/2013/02/08"
Personal tools