Biotech 2nd Edition Sample Ch. 4 Biotechnology Sample Ch. 4 - Page 30

Laboratory 4k Estimating the Amount of DNA in a Sample Using Agarose Gels Background It is helpful to be able to estimate the amount of DNA in an experimental sample. Several methods may be used to detect DNA and estimate its concentration or mass in a sample. If there is enough sample volume, the DNA concentration may be estimated using UV spectrophotometry (see in Chapter 7.) Indicator testing, such as that done with the NUCLEIC dotMETRIC™ kit, allows for estimation of the mass of DNA in a sample. Gel electrophoresis may also be used to estimate or quantify DNA. Running a few microliters of a DNA on an agarose gel can give an estimate of the amount of DNA in a sample. If samples of known concentrations (standards) are loaded onto a gel at a specified volume, then the mass of DNA loaded in those samples will be known. Running unknown samples of the same volume on agarose gels at the same time as the standard samples give a method of estimating the amounts of DNA (mass and concentration) in the unknown samples by comparing the glowing in the stained gel. Purpose To prepare a serial dilution of a known sample of DNA for use as a concentration standard. What is the amount of DNA, measured in µg, in an unknown sample? What is the smallest amount of DNA, measured in µg, visible (resolved) on a 0.8% agarose gel? Materials Microcentrifuge Gel box, horizontal, for agarose gels Prepared agarose gel (from Lab 4i) Electrophoresis Buffer concentrate, TAE (40X or 50X) or LB buffer (10X or 20X) Beakers, 600 mL Tube rack for 1.7-mL tubes Reaction tubes, 1.7 mL Permanent lab marker pens DNA samples (spooled Lambda DNA (uncut), 500ng/ μL, 2 tubes for 16 lab groups DNA loading dye, 10X (for TAE gels) or 5X LB loading dye (for LB gels) Micropipet, P-10 Micropipet, P-100 Micropipet tips for P-10 Micropipet tips for P-100 Lambda/HindIII DNA sizing standard markers, 50 ng/μL (pre-mixed 90 μL sample + 10 μL of 10X DNA loading dye for TAE gels or 20 µL of 5X LB loading dye for LB gels Power supply, 300V Glasses, safety, plastic Weigh boat, 5.5”X 5.5” DNA gel stain (ethidium bromide, 0.5 μg/mL or 1X LabSafe Nucleic Acid Stain™) Deionized water Gel photo imaging system Gloves, large Procedure Note: This activity assumes prior knowledge and experience preparing and running agarose gels. Refer to Lab 4i and Lab 4j if you need help preparing electrophoresis buffer or agarose gels or with loading, running, staining or imaging an agarose gel. 1. Prepare enough 1X electrophoresis buffer to prepare and run a gel. Record the calculations and a diagram to show how you prepared the buffer. Are you using TAE buffer or LB buffer? Find the correct DNA loading dye to use in step 4. 2. Prepare 0.8% agarose in 1X electrophoresis buffer with either 1 row of 8 wells or 2 rows of 6 wells. Pour a gel that is 6–7 mm thick. Leave to cool, undisturbed for 15 min. 3. Prepare four DNA samples at different concentrations to use as concentration standard