Fluorescent Proteins & Plasmid Purification for Optical Microscopy

1. Fluorescent Proteins& Plasmid Purificationfor Optical Microscopy Kristen Downs University of Florida Mentor: Mike Davidson NHMFL REU 2005

2. Materials: QIAGEN Endofree Plasmid Maxi kit Purpose: Prepare up to 500 μg plasmid DNA Bacteria Colony  Plasmid DNA Pellet  Fluorescent Organelles Fluorescent Proteins from Plasmid Purification http://micro/primer/techniques/fluorescence/fluorescentproteins/fluorescentproteinshome.html

3. Green fluorescent protein (aequorin) Isolated from jelly fish Aequorea victoria Fluoresces green Important in cell & molecular biology Biosensors Targeting cytoskeletal elements & organelles FRET – Fluorescence Resonance Energy Transfer FRAP – Fluorescence Recovery After Photobleaching Other mutants engineered Cyan fluorescent protein (CFP) Yellow fluorescent protein (GFP) Enhanced fluorescent proteins (E_FP) DsRed Eos Fluorescent Proteins http://faculty.washington.edu/cemills/Aequorea.html http://www.lifesci.ucsb.edu/~biolum/organism/photo.html

4. Selective Plate Bacteria with plasmid DH5-alpha Competent Agar Antibiotic (Kanamycin) Starter culture 5 mL LB media Antibiotic Autoclaved Refrigerated Plasmid Purification:From Petri Dish to Starter Culture • Select colony • Sterile rod • Incubation • 37˚C • 8 hours

5. Bacterial growth 1/500 to 1/1000 dilution of starter culture 100 mL media with antibiotic Incubation overnight Bacteria in log phase Plasmid Purification:Harvesting the Bacteria

6. Resuspension 10 mL Buffer P1 Non-detergent solution Centrifugation Balance before centrifuging 6000 x g 15 minutes 4˚C Plasmid Purification:Centrifugation & Resuspension

7. Lysis 10 mL Buffer P2 Alkaline NaOH-SDS Destruction of bacterial membrane Lysate appears viscous Neutralization 10 mL chilled Buffer P3 Acidic KAc Fluffy white precipitate forms Genomic DNA Proteins Cell debris Plasmid Purification:Lysis & Neutralization

8. Filter Lysate QIAfilter Maxi Cartridge Let precipitate rise to the top Use plunger to filter cell lysate into 50 mL tube WHERE IS THE PLASMID DNA? Endotoxin Removal 2.5 mL Buffer ER Definition of endotoxin: Lipopolysaccharide (LPS) Cell membrane component of Gram-negative bacteria Released when bacterial cells are broken down or die Ice incubation for 30 min. Plasmid Purification:Filter & Remove Endotoxins

9. Column Chromatography Gravity flow Anion-exchange resin Silica beads of 100 μm with hydrophilic surface coating DEAE (diethyaminoethanol) on surface of resin positively charged DNA in solution negatively charged Salt concentration & pH of buffers determine if DNA is bound or elutes from column Chromatography Buffers Equilibrate column 10 mL Buffer QBT Wash column 2 x 30 mL Buffer QC Medium salt – RNA, protein, carbohydrates, & small metabolites elute Elute DNA 15 mL Buffer QN High salt – plasmid DNA elutes Plasmid Purification:Column Chromatography

10. In Isopropanol 10.5 mL Isopropanol 0.7 volumes of isopropanol relative to eluent DNA precipitate glassy In Ethanol 5 mL 70% Ethanol DNA precipitate white & fluffy Plasmid Purification:DNA Precipitation • Centrifugation • Small centrifuge tubes • 15000 x g • 4˚C • Decant supernatant to isolate pellet

11. Dry DNA In Desiccator Transfect cells with DNA Plasmid Purification:Dry & Resuspend… Ready for Use! • Image Cells • Resuspend • Buffer TE • Tris & EDTA • Shake while dissolving

12. 2 ways to make FLUORESCENT cells Staining of cells or tissues with fluorescent dyes Expression of fluorescent proteins Microscopy Confocal Laser Scanning Microscopy Wide Field Fluorescence Imaging Time-lapse Imaging Summary

13. Thank You! • Optical Microscopy Lab • Mike Davidson • Eric Clark • Greg Ottenberg • Nathan Claxton • Andy Miller • Graphic Artists • Adam Rainey • Lionel Parsons • Matt Crane • Matt Boles • CIRL Staff • Pat Dixon • Gina LaFrazza • Carlos Villa • Stacy Vanderlaan • Sarah Mullins • NHMFL • NSF