The printable version is no longer supported and may have rendering errors. Please update your browser bookmarks and please use the default browser print function instead.
 Building a Reporter Gene
|
<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>
|
February 8, 2013
- Assembly: (5xGal4 : [Spacer : HSVtk TATA]) : (K : AmCyan : AmCyan : NLS : STOP : [polyA2]) (KAH201)/(KAH182); AmCyan reporter gene for chromatin proteins.
Assemblies
- KAH201/KAH182: (1) KAH201/(E/S)/203 + (2) KAH182/(E/X)/1675
> Digests (Fermentas FD)
| Reagent |
Volume |
|
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
|
Building a Reporter Gene
