Dean-Wilder-2011-File-S2-final-1.doc

FILE S2 Laboratory Exercise Supplies for “Frankenplasmid Lab” While the MATERIALS AND METHODS section gives an overview of student and staff procedures, and FILE S1 describes the student protocol in detail, this file lists the supplies that the staff must prepare to implement the module in their classroom. Unless otherwise noted, chemicals were purchased from Sigma-Aldrich Corporation. Abbreviations with quotes indicate how a tube is labeled in the laboratory manual (FILE S1). Liquid volumes represent the minimum volume needed for a pair of students. We strongly recommend giving students an excess of each solution in case an error is made or they overpipet from their supply. We also keep extra hardware supplies on hand (e.g. microcentrifuge tubes). See MATERIALS AND METHODS for further details about the preparation of competent cells, plasmids, backup cultures, and PCR primers. PRELABORATORY EXERCISE 1 Students are to complete this exercise before Week 1. They will need their laboratory manual (FILE S1) and a computer with internet access. WEEK 1, DAY 1 Each pair of students receives: -A set of three Pipetman micropipettors (Gilson; P1000, P200, and P20) with compatible tips -Fine-tipped, permanent marker to label Eppendorf tubes and Petri plates -Vortex Mixer (Fisher Scientific) -2 LB-agar/kanamycin/tetracycline/X-gal plates (see “SOLUTIONS” below) -Bunsen burner or ethanol lamp -Lighter or matches -Bacterial spreader and beaker filled with 70% ethanol for spreader sterilization -Ice bucket containing: pUC-Kan1 or pUC-Kan2 (100 ng/μl in sterile ddH2O) 29 μl pBR322 (New England Biolabs, diluted to 100 ng/μl in sterile ddH20) 29 μl 10X FastDigest Green Buffer (“EB”; Fermentas) 18 μl FastDigest EcoRI (“E”; Fermentas, #FD0274) 4 μl FastDigest HindIII (“H”; Fermentas, #FD0504) 4 μl 10X T4 DNA ligase buffer (“LigB”; Fermentas) 3 μl 2 tubes of competent cells (“CC”) 100 μl/tube Positive control plasmid (for “F” or “G” controls) 5 μl