SEED/2009/Day 3
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Morning
- each group will miniprep 1.5ml of pSB4A5.I52001 and pSB1A3.E0050 cultures
- elute in 30ul
Afternoon
- spec miniprep concentrations
- Set up one 50ul digest of pSB4A5 with EcoRI and PstI
- 5ul NEB2
- 0.5ul BSA
- 2.5ug pSB4A5 miniprep
- 1ul EcoRI
- 1ul PstI
- leave in 37C incubator overnight
Quiz
- Restriction digest and gel electrophoresis
Lecture
- What are you going to learn and do in this class?
- Cloning Process Overview
- Characterization
Instructor Preparation
- grow up cultures of pSB4A5.I52001 (destination vector) and 1A3.E0050 (PCR template)
- prepare minipreps in case student prep concentrations are too low
Instructor Post-prep
- heat kill and store miniprep digests in freezer