Bacteria Transformation

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Bacterial Transformation

1. Take and aliquot of RbCl2 competent cells and thaw on ice

2. Add 1μL of DNA or 10ul of Ligase Reaction

3. Incubate on ice for 30 min

4. Heat shock at 42°C for 30s, move immediately to ice

5. Add 0.5ml of TB media

6. Shake for 30 min at 37°C

7. Plate on LB or TB plates with correct antibiotic; grow in 37°C overnight

8. Add one colony to 2ml of TB/Amp Liquid Culture

9. Grow at 37°C till culture is at saturation

Mini-Prep (See QIAgen Kit Protocol)

QIAgen Miniprep Kit Bench Protocol

1. After the mini-prep digest 1ul of plasmid

20μL Digestion:

1μL Plasmid
2μL of NEBuffer
2μL 10x BSA (if required)
1μL of Restriction Enzyme (if more than 1 enzyme, than use 0.5 μL of each enzyme)
Remaining μL are ddH20

2. Digest the plasmid for 1 hour

3. Use gel electrophoresis to determine whether the correct plasmid has been miniprepped.