220
- Thermal cycler.
- Microcentrifuge machine.
- Gel imager/transilluminator.
- Gel cutter.
- Gel cleanup kit.
- Dry bath.
- Nanospectrophotometer.
- Sanger sequencing reaction kit.
- Purifying sequencing reaction kit.
- DNase-free water.
- Primer set.
- Micropipettes (10, 20, 200, and 1000 μL).
- DNase-free filtered tips (10, 20, 200, and 1000 μL).
- Microcentrifuge machine.
- Thermal cycler.
- Vortexer.
- Reagent reservoir.
- Highly deionized formamide.
- Genetic analyzer.
3 Methods
Refer to Chapter 18 , Subheading 3.1.- All samples are diluted with DNase-free water only.
- Prepare all PCR materials within the PCR hood to reduce
chances of contamination (see Note 1). - Thaw all PCR required ingredients on ice.
- Briefly tap and spin down each reagent to ensure homogeneity.
- Prepare a master reaction mixture (see Note 2) according to
the manufacturer’s protocol with consideration of pipetting
error (see Note 3). - A minimal of two technical replicates per sample (see Note 4)
is required. - For example, for three biological samples, eight master reac-
tion mixtures will be prepared (see Note 5). - Then for each individual reaction, aliquot a single reaction of
master reaction mixture into a PCR tube, and add the tem-
plate (see Note 6) to it.
2.3 Sanger
Sequencing
3.1 Genomic DNA
Extraction from
Clinical Samples
3.2 Targeted Gene
PCR
Katherine T. W. Lee et al.