BIO203 Methods in Molecular Biology

Contents

S. No. Practical

P. No. Preparation of stock and working solutions Isolation of DNA (Part I) Isolation of DNA (Part II) Quantification of DNA by Gel Electrophoresis Isolation of RNA Quantification of RNA by Nanodrop Restriction digestion of DNA and preparation of restriction maps Polymerase Chain Reaction (PCR) Types of PCR and Application Detection of mutations by Restriction Fragment Length Polymorphism (RFLP) Preparation of Chemically Competent Cells Transformation of bacteria with plasmid DNA Isolation of bacterial Plasmid Analysis of proteins by SDS-PAGE

Preparation of Reagents

The following general instructions are applicable in the preparation of all reagents. Use graduated cylinders or pipettes closest to the volume being measured for preparing liquid reagents. Store all reagents in sterile containers unless otherwise noted. Label all reagents with name of reagent, date prepared, initials of scientist that prepared reagent, lot number, and expiration date. Record each preparation in the lab’s reagent logbook.

1M Tris-HCl (Tris Hydroxymethyl amino methane) pH 8

H2O to 800ml Adjust to desired pH with concentrated HCl. Mix and add H2O to 1 Liter.

0.5 M EDTA (Ethylenediamine Tetra acetic Acid) pH 8.0

Na2EDTA.2H2O 186.1g H2O to 700ml Adjust pH to 8.0 with 10M NaOH (almost 50ml) Mix and add H2O to 1 Liter.

10M NaOH

NaOH 400 g H2O to 1 Liter Store at room temperature

10 mg/ml Ethidium Bromide

Ethidium Bromide 0.2 g H2O to 20ml Mix well and store at 4oC in dark

TE (Tris 10 mM-EDTA 2mM) pH 8.0 (Lysis Buffer)

1. 5 M EDTA pH 8.0 4 ml

Low TE (Tris 10 mM-EDTA 0.2 mM) pH 8.0 for DNA storage

1. 5 M EDTA pH 8.0 0.4 ml

H2O to 1 Lite

Proteinase K (10mg/ml)

Proteinase K 100 mg lyophilized powder in Ultra-pure H2O to 10 ml. Make the aliquot and store at approximately -20 C

TEN buffer (10mM Tris, 2mM EDTA, 400 mM NaCl)

1 M Tris-HCl p H 8.0 10 ml 5M NaCl 80 ml

1. 5M EDTA 4 ml

SDS 10% w/v

Sodium dodecyl sulfate 100g H2O to 700ml Heat to approximately 65oC to dissolve. Bring to a final volume of 1.0 L with ultra-pure water.

CAUTION: SDS can be irritating to mucous membranes. Wear safety glasses, mask and gloves when handling

50x TAE (Tris-Acetate-EDTA) Electrophoresis Stock buffer

Glacial acetic acid 57.1 ml

1. 5 M EDTA pH 8.0 100ml H2O to 1 Liter

1x TAE (Tris 40mM, Acetate 20mM, EDTA 2mM) Electrophoresis working buffer

50x TAE 10 ml H2O to 500 ml the pH of diluted buffer is 8.3.