November 6, 2012
- Restriction digests of H2B-GFP and LOV
Restriction Digest Reagents
Reagent
|
H2B-GFP/EcoRI
|
H2B-GFP/PstI
|
LOV/PstI
|
DNA(plasmid)
|
2.0
|
2.0
|
2.0
|
10x buffer
|
1.5
|
1.5
|
1.5
|
EcoRI
|
1.0
|
0
|
0
|
PstI
|
0
|
1.0
|
1.0
|
dH2O
|
10.5
|
10.5
|
10.5
|
Total
|
15.0
|
15.0
|
15.0
|
Expected: 1. H2B-GFP/ EcoRI = 4885, 210 2. H2B-GFP/ PstI = 5101 3. LOV/PstI = 251
|
|
Results of the digests are shown.
- ---Karmella 16:01, 7 November 2012 (EST): Note: Please re-do LOV/PstI
- ---Karmella 16:01, 7 November 2012 (EST): Here is a reaction table for PCR
- First, resuspend dry primer oligos (in blue-capped tubes) to 100 μM
- Then make working solutions of 10 μM
- Reactions:
- H2B-GFP plasmid + BB_H2B fwd + BB_H2B rev
- LOV plasmid + BB_LOV fwd + BB_LOV rev
Reagents |
H2B |
LOV
|
Plasmid DNA |
0.5 μL |
0.5
|
primer 1 (10 μM) |
1.0 |
1.0
|
primer 2 (10 μM) |
1.0 |
1.0
|
2x GoTaq mix |
12.5 |
12.5
|
dH2O |
10.5 |
10.5
|
PCR program:
- 95°C, 3 min.
- [95°C, 30 sec; 57°C, 30 sec.; 72°C, 30 sec.] x35 cycles
- 72°C, 3 min.
- 4°C ∞
|