Lysis cassette
Digestion of Quickchange
| Name: Theo
| Date: 23.07.2011
|
| Continue from Experiment 22.07.2011
Quickchange V.3 with correct Lysis Template
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| Project Name: Quickchange V.3 with correct Lysis Template
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| 5μl
| Buffer, NEB4
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| 5μl
| BSA (10x)
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| 2,5 μl
| Enzym 1
| DpnI
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| 37,5 μl
| DNA
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| Quickchange PCR
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50 μl total volume
Incubate for about 1h at 37°C + Heat inactivation at 80°C for 20min.
Transformation
| Name: Theo
| Date: 23.07.2011
|
| Continue from 23.07.2011 Digestion of Quickchange V.3
Experiment
|
| Project Name: Quickchange V.3 with correct lysis template
|
Procedure
- take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
- thaw cells on ice 20 minutes
- pipette 50 μl cells and 2 μl DNA into eppi still on ice!
- Incubate for 30 minutes on ice
- Heat at 42°C for 60 sec
- Incubate on ice for 5 minutes
- Add 200 μl LB Broth
- Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
- Plate 50 μl and 200μl on two different LB/Agar plates with appropriate antibiotic resistance
Documentation:
Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance etc.
| It was done in ordert to correct the number of nucleotides between RBS and ATG of the temperature sensitive repressor from our Lysis Device, ie to insert 6bp.
This time the correct DNA template was used, ie S11 (K098995). The backbone vector is psB1A3 (Amp resistance)
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