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
|
|
Project name
Main project page
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