Alkaline agarose gel electrophoresis (Sambrook et al., 1989)
Alkaline agarose gels can be used to determine the size and quality of first and second strand cDNA synthesis by reverse transciptase.You will need:
Alkaline loading buffer (300mM NaOH, 6mM EDTA, 18% Ficoll 400, 0.15% bromocresol green, 0.25% xylene cyanol FF)
10 x Alkaline agarose electrophoresis buffer (500mM NaOH, 10mM EDTA, pH 8.0, made fresh for each use)
1) Melt the required amount of powdered agarose in a measured quantity of water, allow to cool to approximately 60°C and add 0.1 volumes 10 x alkaline agarose electrophoresis buffer. Pour gels as described in 'Standard Agarose Electrophoresis'.
N.B: Agarose should not be melted in the presence of NaOH as this causes the hydrolysis of the polysaccharides present in the gel.
2) Samples to be analysed (usually 1-3ul) should be added to 10-20ul 1 x akaline agarose electrophoresis buffer and to this should be added 0.2 volumes of alkaline loading buffer.
3) Load samples and carry out electrophoresis at a maximum of 5V/cm until the bromocresol green has migrated approximately two thirds of the length of the gel.
N.B: It is imperative to prevent the gel and buffer temperature getting above 40-45°C as alkaline hydrolysis of the agarose polysaccharides will occur causing dissolution of the gel. The voltage gradient should therefore be adjusted accordingly. Only gel rigs with a large buffer volume (and therefore a large heat sink) or a cooling system should be run at 5V/cm.
If the samples contain 32P-labelled DNA, after electrophoresis the gels can fixed for 30 minutes in 7% TCA(w/v), dried and subjected to autoradiography.
