IGEM:UC Berkeley/2006/Procedure Overview (Plasmid DNA to Sequencing)

1. Plasmid DNA Path 1 - Restriction Digest of Plasmid DNA - Run a Gel to separate parts - Gel purify the desired part (not always going to be the shorter or longer band- could be either) - Result: Double stranded linear DNA with sticky ends Path 2 - PCR desired Plasmid - Run an analytical gel to determine whether PCR worked correctly - Result is liner double stranded DNA in buffer - PCR cleanup - Result is linear double stranded DNA in H2O - Restriction Digest - Cleanup- take only the strands that we want to insert into plasmid - Result: Double stranded linear DNA with sticky ends

2. Ligation - DNA ligase connects double stranded fragements of DNA with reverse complement sticky ends - Result: Relaxed double stranded circular DNA

3. Transformation- insert new plasmid into cell - Plate - Observe results- are they expected or unusual?

4. Grow up colonies of cells in LB

5. Screen Path 1 - Mini prep - Restriction Map - Result: Indentified clones Path 2 - Colony PCR - Result: Indentified clones

6. Sequence- is plasmid what it should be? --Mfleming 14:34, 3 July 2006 (EDT)