Agarose Gel Electrophoresis

Abstract

This appendix presents a protocol for separating and purifying DNA fragments between 0.5 and 25 kb. A Support Protocol describes the use of midigels and minigels.

Single-Nuclear DNA Instability Analyses by Means of Single-Cell Pulsed- Field Gel Electrophoresis - Technical Problems of the Comet Assay and Their Solutions for Quantitative Measurements

“…Submarine agarose gel electrophoresis is one of the most common types of macro-electrophoresis [41,42] used to determine the size of DNA fragments in the range of 500 to 30,000 base pairs. DNA samples extracted from a cellular population are aligned at the origin and migrate several or more centimeters.…”

Synthesis of Stable Aminoacyl‐tRNA Analogs

“…Below is the procedure to obtain and purify the 74-nt tRNA with a final quantity of about 65 nmol. (Seidman et al, 2001) Liquid and solid LB growth medium (Elbing and Brent, 2002) Ampicillin disodium salt, 100 mg/mL solution (also see Elbing and Brent, 2002) QIAprep Spin Miniprep Kit (Qiagen) Custom-made DNA PCR oligonucleotide primers for amplifying DNA matrix of tRNA sequence (also see Kramer and Coen, 2001): Forward: 5 -TTT AAT ACG ACT CAC TAT AGG GGC CTT AGC TCA GCT GGG AG-3 ) Reverse: 5 -GTG GAG CCT AGC GGG ATC G-3 Agarose, electrophoresis grade (also see Voytas, 2000) 0.7 mg/mL ethidium bromide (also see Voytas, 2000) 10× T7 buffer (see recipe) 25 mM ribonucleotide (NTP) mix (25 mM each ATP, CTP, GTP, and UTP) 1 M MgCl 2 Triton X-100 200 mM guanosine 5 -monophosphate (GMP) T7 RNA polymerase, purified in the laboratory (alternatively can be bought from a wide range of suppliers) DNase I (RQ1 RNase-Free DNase; Promega) Water-saturated phenol, pH 4.5 to 5 (e.g., Eurobio; http://eurobio.free.fr/) 5:1 phenol:chloroform (molecular biology grade; e.g., Sigma) Absolute ethanol (molecular biology grade; e.g., Fisher Scientific), cold 70% ethanol, cold HiLoad 26/60 Superdex 75 prep grad column (GE healthcare) Dex buffer (see recipe)…”

“…PCR thermal cycler (also see Kramer and Coen, 2001) and PCR tubes UV spectrophotometer 0.025 μm nitrocellulose membrane filter, 13-mm diameter (Millipore) Electroporator (also see Seidman et al, 2001) Agarose gel electrophoresis apparatus (also see Voytas, 2000) 1.5-mL plastic reaction tubes Heat block Refrigerated centrifuge FPLC system, with conductivity and 260-nm absorbance detection 0.22-μm syringe filter 15-mL centrifuge tubes (e.g., BD Falcon) Lyophilizer 10. Pick 5 to 10 well isolated colonies growing on either or both plates and transfer bacteria in 5 mL sterile LB medium with 100 μg/mL ampicillin.…”

“…Supplement 44 Current Protocols in Nucleic Acid Chemistry Molecular membrane tubing, MWCO 12,000 to 14,000 (Spectra/Por) Boiling water bath Additional reagents and equipment for the polymerase chain reaction (Kramer and Coen, 2001), spectrophotometric determination of DNA concentration (UNIT 5.2), electroporation (Seidman et al, 2001), preparation of bacteriological media (Elbing and Brent, 2002), agarose gel electrophoresis (Voytas, 2000), denaturing polyacrylamide electrophoresis (Support Protocol 3), and dialysis (Zumstein, 1998) Prepare the DNA matrix 1. Note that the sequence of the tRNA Ala (5 -GGG GCC UUA GCU CAG CUG GGA GAG CGC CUG CUU UGC ACG CAG GAG GUC AGC GGU UCG AUC CCG CUA GGC UCC ACC A −3 ) corresponds to the three identical sequences annotated as tRNA Ala in the genome sequence of E. faecalis strain V583.…”

Diagnosis and Treatment of Mycoplasma‐Contaminated Cell Cultures

“…Transfer each tube to the thermal cycler and perform PCR using the following cycling conditions: 10. Following amplification, resolve the PCR products on a 1% agarose gel (see, e.g., Sambrook and Russell, 2001;Voytas, 2000).…”