November 2, 2012 Digestion and PCR Planning
- Check LOV and H2B-GFP minipreps with digestion
- BioBrick PCR
Minipreps
Measure concentrations
Sample
|
260/280
|
ng/μL
|
LOV
|
1.834
|
281.868
|
H2B-GFP
|
1.87
|
118.217
|
Check miniprep results
- Use a plasmid editor software to check sequences for restriction digest enzymes
- LOV has two PstI sites while H2B-GFP has one PstI, one NotI, and two EcoRI sites
- Decide to cut LOV with PstI and H2B-GFP with PstI and EcoRI
- Expected bands for LOV at 251 with PstI digest
- Expected bands for H2B-GFP at 4885, 205 with Psti and EcoRI digest
Reagent |
LOV |
H2B-GFP/ PstI |
H2B-GFP/ EcoRI
|
DNA(plasmid) |
2.0 μL |
2.0 |
2.0
|
10X buffer |
1.5 |
1.5 |
1.5
|
PstI |
1.0 |
1.0 |
---
|
EcoRI |
--- |
--- |
1.0
|
dH2O |
10.5 |
10.5 |
10.5
|
|
15.0 |
15.0 |
15.0
|
Results
Expected: 1. LOV/PstI = 251 2. H2B-GFP/ PstI = 5101 3. H2B-GFP/ EcoRI = 4885, 210
|
15 μL/lane; 1% agarose
|
|