<p>Solutions (Note: Make sure labels contain the name of the reagent, pH, your name, and the date made on every solution)</p><p>1M Tris – HCL pH 7.5 -Make a solution of 1M Tris (Free Base) in a final volume of 100 ml molecular weight (MW) of Tris = 121.1 g/mol (MW)(desired concentration mol/L)(final volume in L) = grams of reagent -Add half the final volume of diH2O to the powder, mix. -Before bringing the solution to full volume, pH with conc. HCL using a pH meter -Bring solution to full volume -Autoclave</p><p>10% (w/v) SDS -Make a 10% solution of sodium dodecyl sulfate (SDS). Also, called Lauryl Sulfate in a final volume of 50 ml. DO NOT MIX VIGROUSLY, Watch for bubbles! [X (g)/50 ml] * 100% = 10% solution of SDS -Caution: SDS is a dangerous chemical and an irritant. Don’t inhale or touch skin. -Do not autoclave</p><p>Isopropanol (50 ml) -Get from stock bottle -Do not autoclave</p><p>.5M EDTA pH 8.0 -Make a solution of .5M EDTA in a final volume of 100 ml molecular weight (MW) of EDTA = 372.2 g/mol (MW)(desired concentration mol/L)(final volume in L) = grams of reagents -EDTA takes a while to dissolve; need to heat slightly (on low) and add some NaOH -Before bringing the solution to full volume, pH with NaOH using a pH meter -Bring solution to full volume -Autoclave.</p><p>70% (v/v) ethanol (100 ml) -Use 100% ethanol -Do not autoclave.</p><p>10% glycerol (500ml – 1000 ml) -Make from 100% glycerol stock [X/1000ml] * 100% = 10% solution of glycerol -Put everything in a 1L bottle, mix, and autoclave. -Store in bottle at 4C Resuspension Solution (100 ml) -Add Tris-HCl (pH 7.5, [Tris-HCl]i=1M, [Tris-HCl]f=50mM) -Add EDTA (pH 8.0, [EDTA]i=.5M, [EDTA]f=10mM) -Bring to full volume with dH2O -Autoclave</p><p>Lysis Solution -Make fresh each time with 10% SDS, 10N NaOH and dH2O</p><p>Neutralization Solution pH 4.8 -Make a 100 ml solution of 1.32M KOAc (potassium acetate) molecular weight (MW) of KOAc = 98.14 g/mol (MW)(desired concentration mol/L)(final volume in L) = grams of reagents -Before bringing the solution to full volume, pH with acetic acid using a pH meter -Bring solution to full volume -Autoclave</p><p>LB broth -Using a clean 1L bottle, measure 500ml of distilled H2O using a graduated cylinder. -Add 12.5g of Dehydrated LB broth to the bottle. -Autoclave.</p><p>LB Agar -Using a clean 1L bottle, measure out 500ml of distilled H2O using a graduated cylinder. -Add 12.5g of Dehydrated LB broth and 9.0g of Bacto agar to the bottle. -Autoclave.</p><p>SOB media (1000 ml) -make as per Maniatis, Vol3. pg A2 -Add 20g of bacto-tryptone, 5g of bacto-yeast extract, and .5g NaCl to a bottle with 925 ml of dH20. Mix. Add KCl ([KCl]i=250mM, [KCl]f=2.5mM) -Bring to full volume. -Autoclave.</p><p>SOC media (500 ml) -follow directions on SOB (+Mg2+) bottle -Autoclave. -Add glucose ([glucose]i=1M, [glucose]f=20mM) once cooled</p><p>1M Glucose -Make a 100ml solution of 1M glucose molecular weight (MW) of glucose = 180.2 g/mol (MW)(desired concentration mol/L)(final volume in L) = grams of reagents -Autoclave. 10M NaOH -Make a 100ml solution of 10M NaOH -Store in plastic bottle -Add dH2o first molecular weight (MW) of NaOH = 56.11 g/mol (MW)(desired concentration mol/L)(final volume in L) = grams of reagents -Do not autoclave</p><p>250mM KCl -Make a 100ml solution of 250mM KCl -Add dH2o first molecular weight (MW) of KCl = 74.55 g/mol (MW)(desired concentration mol/L)(final volume in L) = grams of reagents -Autoclave</p><p>8.5% (w/v) NaCl -Make a 150ml solution of 8.5% NaCl -Autoclave</p><p>1M Hepes pH 7.4 -Make a 100ml solution of 1M Hepes -Before bringing the solution to full volume, pH with NaOH using a pH meter -Bring solution to full volume -Autoclave</p><p>HN Buffer (100 ml) -Add Hepes (pH 7.4, [Hepes]i=1M, [Hepes]f=20mM) -Add NaCl ([NaCl]i=8.5%, [NaCl]f=.85%) -Bring to full volume with dH2O -Autoclave</p><p>Loading Buffer for Agarose gel electrophoresis (5 ml) -Add .25% (w/v) bromophenol blue, .25% (w/v) xylene cyanol, and 15% (w/v) Ficoll to a beaker. -Add 5ml of dH2O to beaker. Aliquot into microcentrifuge tubes. -Do not autoclave. -Note: 40% (w/v) sucrose or 30% (v/v) glycerol can be use in place of ficoll</p>