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From 2010.igem.org

Contents 1 Nina 1.1 Testing DH5α competent cells by transformation 1.2 Preparation LB agar plates with ampicillin resistance 2 Johan 2.1 Transformation 2.2 LB agar plates with ampicillin 2.3 PCR 2.3.1 Mix 2.3.2 Program

Nina

Testing DH5α competent cells by transformation

This was performed according to tranformation protocol From NEB 5-alpha competent E.coli (High Efficiency) NEW ENGLAND BioLabs.

I transformed both my own cells from yesterday and also the kit's competent cells. Each one had a two serial 10-fold dilution.

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Preparation LB agar plates with ampicillin resistance

LB agar:

• 25 g LB
• 15 g bactoagar
• Fill up to 1 L with distilled water

1. Autoclave for 120°C, 20 min
2. Cool down
3. Add 200 mg ampiciliin in 1,8 ml water
4. Pour out on plates

Johan

Transformation

A transformation was performed to test the competence of the cells, both with our own and competent cells from a kit.

LB agar plates with ampicillin

• 25 g LB
• 15 g bactoAgar
• up to 1L ddH2O

• Autoclave 120 °C 20 min
• Cool down
• Add 200 mg Amp in 1,8 ml H2O
• For 1L of LB add 1 ml of 100 mg/ml

• Pour on plates

PCR

PCR of SOD and yCCS to add prefix/suffix

Mix

• 30 µl H2O
• 10 µl 5x buffer
• 1 µl 50 mM MgCl2
• 1 µl 10 mM dNTPS
• 1 µl DNA templ.
• 3 µl 5 µM F.primer
• 3 µl 5 µM R.primer
• 1 µl PfuX Polymerase

Program

1: 98 °C 2 min

2: 98 °C 10 sec

3: 55 °C 15 sec

4: 72 °C 45 sec

2-4 repeat 30 times

5: 72 °C 5 min

6: 4 °C ∞