Objectives
- Digestion of Plasmid extraction pSB1AK3/BBa_B0015 for Standard Assembly.
- PCR for BBa_P1003 with preffix and suffix primers.
- See in gel digestion and product of PCR.
- Clean today´s PCR product.
- See gel to perform double digestion.
- Overnight double digestion with EcoRI and SpeI for PCR purified.
Digestion
- Digestion of plasmid pSB1AK3/BBa_B0015 with EcoRI and XbaI. 8 hrs, 37°C.
| Reagents
| μL
|
| H2O
| 5
|
| Buffer EcoRI NEB
| 6
|
| BSA 10X
| 6
|
| DNA pSB1AK3/BBa_B0015
| 40
|
| EcoRI NEB
| 1.5
|
| XbaI NEB
| 1.5
|
Enzymes inactivation= 20 min 65°C
PCR for BBa_P1003
| Reactives
| Tube 1
| Tube 2
|
| H2O
| 31.5
| 31.5
|
| Buffer Taq Standard
| 5
| 5
|
| dNTPs 8mM
| 1.25(.2mM)
| 1.25(.2mM)
|
| DNA Paz's PCR BBa_P1003
| 1
| 1
|
| Oligo F
| 5
| 5
|
| Oligo R
| 5
| 5
|
| Taq Standard
| 1.25
| 1.25
|
| Total
| 50
| 50
|
Initialization step: 3:00min 95°C
Denaturation step: 30seg 94°C
Annealing step: 30seg 55°C
Extension/elongation step: 40seg 72°C
Final elongation 30seg 72°C
Final hold: 3hrs 4°C
Gel of PCR and digestion
- 1 Gene'O Mixed Ruler
- 2 Paz's PCR ~1Kbp as expected
- 3 Tube 1 PCR ~1Kbp as expected
- 4 Tube 2 PCR ~1Kbp as expected
- 5 Dobuble Digestion ~3.4Kbp as expected
PCR product purification
Roche´s kit.
Final dilution= 60μL
Gel of PCR purified Vs Plasmid Cut
This gel just show concentration between backbone and insert and concentration for Double Digestion of insert(Purified PCR product)
Double Digestion of PCR product
Double Digestion with EcoRI-HF and SpeI
| Reagents
| μL
|
| Buffer4 10x
| 6
|
| BSA 10x
| 6
|
| PCR product(BBa_P1003)
| 40
|
| SpeI
| 1
|
| EcoRI-HF
| 1
|
| H2O
| 6
|
| Total
| 60
|
Status
Success!!!
|
iGEM 2011
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