Solutions (Note: Make sure labels contain the name of the reagent, pH, your name, and the
date made on every solution)
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
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
Isopropanol (50 ml)
-Get from stock bottle
-Do not autoclave
.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.
70% (v/v) ethanol (100 ml)
-Use 100% ethanol
-Do not autoclave.
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
Lysis Solution
-Make fresh each time with 10% SDS, 10N NaOH and dH2O
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
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.
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.
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.
SOC media (500 ml)
-follow directions on SOB (+Mg2+) bottle
-Autoclave.
-Add glucose ([glucose]i=1M, [glucose]f=20mM) once cooled
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
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
8.5% (w/v) NaCl
-Make a 150ml solution of 8.5% NaCl
-Autoclave
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
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
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