Restriction Enzyme Test
- To test restriction enzymes, digest DNA with E+P, X+S, E+S, X+P; in addition to this, do four single cuts.
- We can tell which enzyme is not working (if it is such) by examining which pairs are not cutting properly.
- I will cut Jason's miniprepped DNA because it had the most amount of DNA present.
- 4 double digests + 4 single cuts, for further confirmation.
- Volumes used:
| Reagent
|
Control
|
E
|
X
|
S
|
P
|
ES
|
EP
|
XS
|
XP
|
Master Mix
|
| 10X BSA |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
20
|
| 10X Buffer 2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
20
|
| EcoRI |
0 |
1 |
0 |
0 |
0 |
1 |
1 |
0 |
0 |
N/A
|
| XbaI |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
N/A
|
| SpeI |
0 |
0 |
0 |
1 |
0 |
1 |
0 |
1 |
0 |
N/A
|
| PstI |
0 |
0 |
0 |
0 |
1 |
0 |
1 |
0 |
1 |
N/A
|
| DNA |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
30
|
| sdW |
30 |
30 |
30 |
30 |
30 |
30 |
30 |
30 |
30 |
300
|
| Total |
37 |
38 |
38 |
38 |
38 |
39 |
39 |
39 |
39 |
370
|
- Digest for ~1-2 hrs (not so crucial) at room temp.
- Note: Initially wanted only 10 µl of water, but in the end, I calculated for 30 µl instead in the master mix for a total of 300 µl of water. Therefore, the reaction may be a bit dilute, which is why I will let this run while I continue writing and rehearsing the podcast instead. The timing shouldn't be too important.
- Digest ran overnight on ice after ~2h incubation in 37ºC.
|
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