Lab Safety Gloves (optional) and safety glasses (yes) Labcoats – optional, buy your own Using pipettemen (small volume pipetting) Other pipettes (larger volumes) Gels: agarose vs polyacrylamide; resolution running buffers and loading buffers visualizing DNA Lab exercise; pouring gel, mixing samples and running gel

What is BSL1? http://www.safety.duke.edu/safetymanuals/Lab/default.htm

Starch gel electrophoresis Separation of proteins by size Resolution not so good Smithies, O., Zone electrophoresis in starch gels: group variations in the serum proteins of normal human adults (1955) Biochem J 61: 629 Oliver Smithies

agarose vs polyacrylamide For separation of DNA/RNA and protein molecules by size Resolution – agarose gels, usually 0.7 – 2.0 % horizontal gels 50 bp to 10 -15 kb Higher the concentration – better resolution of smaller sizes Lower the concentration – better resolution of larger sizes Higher concentration – longer runtime Voltage 50 -100 V common, heat can be an issue (melted gels/equipment) RNA gels also made with agarose PAGE (polyacrylamide gel electrophoresis) vertical gels Polyacrylamide gels – base pair resolution Sequencing gel 6% gel 19:1 acrylamide:bis-acrylamide + 6 M urea Protein PAGE gels also, denaturing/non-denaturing

Setting up an agarose gel Put combs in gel mold, dams if needed Measure out buffer (120 ml) into flask Add agarose (1.2 g, ~1 % gel) Boil (hot plate or microwave) Cool to touch Pour and wait to solidify Remove combs, gently Add buffer sufficient to cover gel Load samples, run Stain ~ 5 min with EtBr ( 2 mg/ml) Visualize with UV light source in dark

Prepare a 5X stock solution in 1 L of H2O: 54 g of Tris base TBE buffer Prepare a 5X stock solution in 1 L of H2O: 54 g of Tris base 27.5 g of boric acid 20 mL of 0.5 M EDTA (pH 8.0) The 0.5X working solution is 45 mM Tris-borate/1 mM EDTA. $5.39 for 76 oz Borax gel prep 10 X stock of borax (100 mM) 19 g of borax in 500 ml, resulting pH 9.3 Assuming Na2B4O7·10H2O, MW= 381.37 Use 1X (10 mM) for agarose gel and running buffer (pH 9.0) Based on BioTechniques 36:214 76 oz = 2.1 kg Enough for 56 liters of 10X stock = 560 liters of buffer ~11200 50 ml gels @ 0.05c/gel 50X TAE (1 liter @ $50) from Boston Bioproducts = 50 liters of buffer ~ 1000 50 ml gels @ 5c/gel

DNA Markers Ladders (1 kb, 100 bp, etc) 1 kb ladder (Boston Bioproducts) Buffered/EDTA solution of high range DNA ladder (10, 8, 6, 5, 4, 3, 2, and 1, 0 kb) Restriction digests of genomes (plasmids or phage primarily) 1 kb DNA ladder

6X gel loading buffer Bromophenol blue 0.25%, Xylene cyanol 0.25%, and Glycerol 40% in autoclaved DI water Can also use sucrose for density Can leave out BPB or XC Xylene cyanol – size marker 10,000 bp at 0.7% agarose 4000 bp at 1.5% 750 bp at 2.0% 200 bp at 3.0% Bromophenol blue – size marker 1000 bp at 0.7% agarose 500bp at 1% 150bp at 2% 50bp at 3%

Many options EtBr, SYBR Green, GelRed,… Staining the gel Many options EtBr, SYBR Green, GelRed,… Ethidium bromide (EtBr) intercalating dye 10 mg/ml (Sigma, BB, others; cheap) 5 ml > 25 ml running buffer = 2 mg/ml working solution

1 kb ladder 15 ml Lambda genome DNA, uncut 10 ml N. crassa genome DNA, uncut 10 ml pUC19 10 ml pBSSK+ 10 ml pGREEN 10 ml pCRISPR 10 ml