Team:HokkaidoU Japan/Notebook/September6
From 2010.igem.org
Ligation of pSB1C3 PCRed from 1-3A and 1-5A(RFP reporter)
- Purified through gel extraction to remove primers
- Digested as listed in the table below
Reagent | Amount |
---|---|
RFP (1-5A) | 0.75 uL |
DW | 34.3 uL |
0.1% BSA | 5 uL |
10x M Buffer | 5 uL |
EcoR I | 2.5 uL |
Pst I | 2.5 uL |
Total | 50 uL |
Reagent | Amount |
---|---|
pSB1C3 | 0.75 uL |
DW | 34.3 uL |
0.1% BSA | 5 uL |
10x M Buffer | 5 uL |
EcoR I | 2.5 uL |
Pst I | 2.5 uL |
Total | 50 uL |
Reagent | Amount |
---|---|
RFP (1-5A) | 0.75 uL |
DW | 34.3 uL |
0.1% BSA | 5 uL |
10x H Buffer | 5 uL |
EcoR I | 2.5 uL |
Pst I | 2.5 uL |
Total | 50 uL |
Reagent | Amount |
---|---|
pSB1C3 | 0.75 uL |
DW | 34.3 uL |
0.1% BSA | 5 uL |
10x H Buffer | 5 uL |
EcoR I | 2.5 uL |
Pst I | 2.5 uL |
Total | 50 uL |
- Extracted from filter with 25 uL, 50 uL DNase free water
- At this point solution volumes were 22.56 ng/uL,45.12 ng/uL acordingly
- Added 5 uL Mighty Mix and 0.5 uL T4 ligase to pSB1C3 2 uL, and RFP 3 uL
- Ligated
- Transformed to 50 uL of competent cells