Team:Pitt/Skin Probiotic/Cathelicidin/Methods

Methods for Cathelicidin

DNA Digest with Thermo Scientific FastDigest Restriction Enzymes
1.1. Digest Insert with XbaI and PstI or EcoRI and SpeI
1.2. Digest Vector Backbone with SpeI and PstI or EcoRI and XbaI
Gel Electrophoresis
2.1. Create 1% agarose gel with EtBr
2.2. Load gel lanes with Invitrogen 1kb Plus ladder and restriction digest products
2.3. Run Gel until band separation is visible
Gel Extraction and Purification
3.1. Image the gel and extract the desired bands
3.2. Perform Gel Purification using Promega SV Wizard Gel and PCR Clean Up System
3.3. Measure DNA concentration using Fisher Scientific Nanodrop 2000
Ligation of Vector Backbone and Insert with T4 DNA Ligase
Transform Ligation Products into Mach1 Competent Cells
Spread Transformation Mix onto selection plate
6.1. Selected on either Ampicillin or Chloramphenicol depending on resistance gene
6.2. Incubate overnight
Select colony from plate and create 5 mL liquid cultures
7.1. Incubate 5-8 hours
Extract Plasmids using Qiagen Miniprep Kit
8.1. Measure DNA concentration using Fisher Scientific Nanodrop 2000
Diagnostic DNA Digest
9.1. Digest Plasmids with XbaI and PstI
9.2. Run Digestion products on 1% agarose gel with EtBr
9.3. Check for correct band lengths