8/17/2010
12:30PM
Gel of PCR from 8/16
1% Agarose gel run at 150V for ~25min.
Lane
1. 1kb ladder
2. LamB EcoRI (5uL + 1uL dye)
3. LamB EcoRI (5uL + 1uL dye)
4. (-) Control (no template)
1:00PM
ArsR Re-Streak
2nd streak of ISB207 ArsR SDM2 pSB1A2
1:30PM
PCR Purification
PCR Purification of LamB EcoRI using Qiagen Kit
2:00PM
Digestion
Digesting LamB and pSB1C3 + RFP with EcoRI and SpeI
| |
LamB PCR Product |
pSB1C3 + RFP |
| DNA |
10uL |
1 μL |
| H2O |
32.5uL |
41.5uL |
| Buffer 4 (NEB) |
5uL |
5uL |
| BSA |
0.5uL |
0.5uL |
| EcoRI |
1uL |
1uL |
| SpeI |
1uL |
1uL |
37C Incubation for 15min.
80C Inactivation for 20min.
3:30PM
Ligation
Ligating pSB1C3 backbone with LamB using EcoRI and SpeI ends
| pSB1C3 + LamB |
| H2O | 13uL |
| pSB1C3 (cut with EcoRI, SpeI) | 2uL |
| LamB (cut with EcoRI, SpeI) | 2uL |
| T4 DNA Ligase Buffer | 2uL |
| T4 DNA Ligase | 1uL |
Room Temperature incubation for 10min.
42C for 20min -- Oops.
80C inactivation for 20min.
4:30
Transformation - Electroporation
Thaw cells --> +50uL cells + 4uL Ligation mixture --> Electroporate (The machine said Arc) --> +1mL SOC medium --> 37C recovery for 1hour.
Samples:
Control pUC19 Amp resistant plasmid
LamB pSB1C3 ligation
Plating
300uL on pre-warmed Cap and Amp plate.
| 
UIUC - Arsenic Collection System!
Main project page
Previous entry Next entry
