Team:University College London/Week11Yanika
From 2012.igem.org
The first digest was carried out in order to prepare the Upstream Part for ligation through an EcoRI-HF™ and SpeI preparative digest:
Ingredient | Amount |
---|---|
Upstream Part Plasmid | 500 ng |
EcoRI-HF | 1 µl |
Spel | 1 µl |
10X NEBuffer 2 | 5 µl |
100X BSA | 0.5 µl |
H2O | to 50 µl |
Another digest was carried out in order to prepare the downstream DNA for ligation through an XbaI/PstI preparative digest:
Ingredient | Amount |
---|---|
Downstream Part Plasmid | 500 ng |
Xbal | 1 µl |
Pstl | 1 µl |
10X NEBuffer 2 | 5 µl |
100X BSA | 0.5 µl |
H2O | to 50 µl |
Another digest was carried out to prepare the backbone plasmid through an EcoRI-HF/PstI preparative digest:
Ingredient | Amount |
---|---|
Destination Plasmid | 500 ng |
EcoRI-HF | 1 µl |
PstI | 1 µl |
10X NEBuffer | 2.5 µl |
100X BSA | 0.5 µl |
H2O | to 50 µl |
For each of the digests above, incubate all three restriction digest reactions at 37°C for 10 minutes and then heat inactivate at 80°C for 20 minutes.
Ligate the Upstream and Downstream Parts into the digested Destination Plasmid, using the following:
Ingredient | Amount |
---|---|
Upstream Part digestion | 2 µl |
Destination Plasmid digestion | 2 µl |
10X T4 DNA Ligase Buffer | 2 µl |
T4 DNA Ligase | 2 µl |
H2O | 11 µl |
After this is prepared, incubate at room temperature for 10 minutes and then heat inactivate at 80°C for 20 minutes.