mm/dd/yyyy
Calculated how many ul of water to add to the gel extractions to be at 20fmol/ul for golden gate reactions:
Sample
|
ng/µL
|
280
|
ug DNA
|
fmol
|
ul to add to diltute to 20fm/ul
|
G GG mCh |
37.513 |
0.022 |
1.87565 |
4740 |
187
|
G GG I13522 no GFP 2 |
47.261 |
0.028 |
2.36305 |
1790 |
39.5
|
G GG I13522 no GFP 3 |
32.562 |
0.02 |
1.6281 |
1230 |
11.5
|
G GG I13522 no GFP 4 |
45.351 |
0.029 |
2.26755 |
1720 |
36
|
G GG I13522 no GFP 5 |
40.521 |
0.024 |
2.02605 |
1530 |
26.5
|
After diluting, set up 4 reactions following Dana's Golden Gate protocol.
Reaction:
Ingredient
|
Volume
|
Bbone at 20fm/ul |
1ul
|
Insert 1 at 20fm/ul |
1ul
|
10X NEB T4 ligase buffer |
1ul
|
NEB T4 ligase (400U/ul) |
1.25ul
|
BsmBI |
0.5ul
|
Water |
5.25ul
|
Rxn #
|
Backbone
|
Insert 1
|
1 |
G GG I13522 no GFP 2 |
G GG mCh
|
2 |
G GG I13522 no GFP 3 |
G GG mCh
|
3 |
G GG I13522 no GFP 4 |
G GG mCh
|
4 |
G GG I13522 no GFP 5 |
G GG mCh
|
Thermocycle
- Cycle 25X
- 45°C 2min
- 16°C 5min
- 60°C 10min
- 80°C 20min
|