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Extracting RNA from Tissue
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Solutions
Buffer (10x) for enzyme reaction (normally comes with the enzyme)
50 mM magnesium chloride
1 mg/ml acetylated bovine serum albumin (BSA)
0.1 M dithiothreitol
Gel loading buffer (5x)
10x Taq polymerase buffer
This is normally supplied by the manufacturer and works very efficiently in the PCRs described here. The usual constituents are:
200 mM TRIS-HCl (pH 8.3)
500 mM KCl
15 mM MgCl2
Dithiothreitol (0.1M) (DTT)
If the reverse transcriptase buffer is supplied with the enzyme this will contain DTT or a 0.1 M DTT stock will be also be supplied. If not prepare a 0.1 M solution in sterile distilled water and store in 100 µl aliquots at -20°C.
dNTP solution
If the individual deoxynucleotides are purchased as solids they must be dissolved in sterile distilled water and carefully neutralised with sodium hydroxide, or they will not be stable. It is therefore recommended to buy them as 100 mM stock solutions which are available from chemical suppliers. These are mixed together and diluted to give a stock solution containing 10 mM each of dATP, dCTP, dGTP and dTTP (i.e., 10 µl of each dNTP and 60 µl sterile distilled water).
10x TRIS-borate buffer (TBE)
109 g TRIS base
55 g boric acid
9.3 g EDTA
Make up to 1 litre with distilled water and check that the pH is 8.3.
20x TRIS-acetate buffer (TAE)
96.8 g TRIS base
22.85 ml glacial acetic acid
40 ml 0.5 M EDTA (pH 8.0).
Make up to 1 litre with distilled water and autoclave.
Ethidium bromide
Dissolve 10 mg in 1 ml sterile distilled water and store at 4°C in a dark bottle. A stock 10 mg/ml solution is generally supplied by companies and this can be diluted 1/10 with distilled water.
TAKE CARE HANDLING THIS
5x Reverse transcriptase buffer
250 mM TRIS-HCl
15 mM MgCl2
38.5 mM KCl
Agarose gels
Normally 1.5-2% gels are used to separate small fragments (less than 500 base pairs) of DNA. For a 2% 50 ml gel weigh out 1.0 g of electrophoresis grade high gelling temperature agarose and add 50 ml of 1x TBE. Melt the agarose (preferably in a microwave oven), cool to 50-60°C, add 10 µl 1.0 mg/ml ethidium bromide solution, mix and pour into the gel former. A more expensive form of agarose, 'Nusieve', can also be used. It has the advantage that when 3% gels are made the background is lower when the gel is stained with ethidium bromide. However, the cheaper alternative is suitable for most purposes.
WARNING - If a microwave oven is not available then the agarose should be heated in a bath of boiling water to melt it. DO NOT place it directly on a hotplate. Care should be taken when melting and handling molten agarose. Wear protective gloves when handling the flask containing the molten agarose and take care not to come into contact with the ethidium bromide.
5x Agarose gel loading buffer
Ready made buffer can be purchased from several companies
20% Ficoll 400 (or 40% sucrose or 30% glycerol)
25 mM EDTA
0.05% bromophenol blue
0.03% xylene cyanol
For 5ml:
1.0 g Ficoll (or 2 g sucrose or 1.5 ml glycerol)
250 µl 0.5M EDTA (pH 8.0)
50 µl 5% bromophenol blue
50 µl 3% xylene cyanol
Make up to final volume of 5 ml with distilled water.
EDTA (0.5M)
Weigh out the appropriate amount of EDTA to make a 0.5M solution and dissolve in 90% of the required volume of water. Adjust pH to 8.0 with sodium hydroxide pellets before making up to the final volume. Note: EDTA will not dissolve completely until the pH is near 8.0.
DNA molecular weight markers
Use either 100 or 123 base pair ladders.
Dilute 0.5 µl stock (1 µg/µl) in 7-10 µl of 1x agarose gel loading buffer for each well.
Primers
Dissolve primer stocks at 100 pmol/l in RNAse-free water and store at -20°C. Make up a working stock of 10 pmol/l.
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