一篇整理的比较好的关于核酸定量的文章
丁香园论坛
1206
DNA QUANTITATION
--------------------------------------------------------------------------------
AGAROSE-GEL METHOD WITH ETHIDIUM BROMIDE STAINING
1) The DNA for assessment was removed from cold storage and heated at 37oC for 30 min (or 3 min at 60oC ). Flicking the bottom of tubes ensured that all the DNA has been suitably resuspended.
2) The samples were then given a quick spin in a microcentrifuge and kept on ice prior to use.
3) 1µl of the DNA sample + 2µl loading dye + 10 µl sterile distilled water were mixed together.
4) Then additional tubes were set up comprising standard DNA (lambda or calf thymus DNA are routinely used in calibration. In general eukaryotic DNA is best used for eukaryotic samples.)
c. 25 ng/µl 0.5µl calf thymus DNA
(50ug/ml stock) + 2µl loading dye + 10.5µl sterile distilled water
c. 50 ng/µl 1.0µl " + " + 10.0µl "
c. 100 ng/µl 2.0µl " + " + 9.0µl "
c. 200 ng/µl 4.0µl " + " + 7.0µl "
5) These tubes were given a short spin.
6) The samples were loaded with the DNA standards at one end of a row of samples.
7) For a rough guide. Commercially available Lambda Hind III marker is used at 500 ng/ml conc. Heated at 60oC for 3 min, the marker (mixed with a suitable loading dye - see section on MOLECULAR TERMS AND SOLUTIONS) can indicate DNA concentration because its heaviest (20 kb band) approximates to 250 ng/µl of DNA concentration and the next two bands are 125 ng/µl and 67.5 ng/µl respectively.
7) The gel was typically run (at 100v) for 30 to 60 min.
8) The technique above was adapted for use with 6% polyacrylamide gels. 10µl volumes of the samples and standard DNA samples were added to the wells of a horizontal gel and the sample was run for 3h at 200v. After this the gel was stained using a silver stain procedure, described below.
--------------------------------------------------------------------------------
SPECTOROPHOTOMETER-ABSORBANCE ASSAYS
1) The DNA for assessment was removed from cold storage and heated at 37oC for 30 min (or 3 min at 60oC ). Flicking the bottom of tubes ensured that all the DNA has been suitably resuspended.
2) The samples were then given a quick spin in a microcentrifuge and kept on ice prior to use.
3) On older models of the spectrometers, the deuterium lamp was turned on 30 min before analysis to allow the lamp to heat up.
4) 2.00 ml of Filtered TE were aliquoted into 10mm dual light path disposable cuvette (Hughes & Hughes Ltd.) and used to zero the spectrometer = 1st cuvette.
5) In a second disposable cuvette 10µl of the DNA sample was pipette pumped in 1.99 ml of TE = sample cuvette. After zeroing the spectrometer on the 1st cuvette, the sample cuvette was brought into place to obtain an absorbance reading.
6) Readings were taken at wavelengths of 260 nm (OD260) and 280 nm (OD280) (the spectrometer was rezeroed between each wavelength reading).
(260 nm for nucleic acids and 280nm for protein concentrations).
The ratio between the readings (OD260/OD280) provided an estimation of sample purity: pure preparations had values close to 1.8 and protein contaminated samples were significantly lower. OD260 also allowed the calculation of nucleic acid concentrations of the samples, i.e. an OD of 1 corresponded to approximately c.50µg /ml for double stranded DNA.
--------------------------------------------------------------------------------
DNA FLUOROMETRY
1) The DNA for assessment was removed from cold storage and heated at 37oC for 30 min (or 3 min at 60oC ). Flicking the bottom of tubes ensured that all the DNA has been suitably resuspended.
2) The samples were then given a quick spin in a microcentrifuge and kept on ice prior to use.
3) On older models of the fluorometers, the machine was turned on 30 min before analysis or until the read-out had stabilised.
4) (For low concentrations of DNA, a eukaryotic 50 µl stock of 25ng/µl calf thymus DNA was used.)
5) Hoechst stain (1mg/ml) was diluted in 25 ml of 1 x TNE (filtered: 10mM Tris, 1mM EDTA, 0.1M NaCl) to a final concentration of 100ng/ml. The solutions were kept in 25 ml sterile specimen bottles (Granier) which were wrapped in silver foil to protect against light.
6) The fluorometer was zeroed using 2 ml of TNE.
7) The fluorometer was then first standardised using 100ng of DNA stock (25ng/µl) (8µl of standard in 2 mL of TNE). The solution was pipette-pumped using a 1000µl Gilson directly in the crystal cuvette and then the scale setting was adjusted to 100 for subsequent readings.
8) This was then followed by standards of 25 ng (2µl), 50ng (4µl), 75ng (6µl), 125ng (10µl) and 150ng (12 µl) of DNA stock in fresh 2 ml volumes of TNE.
9) Washes of the cuvette were carried out by tipping out the old sample and washing with a 1 ml aliquot of TNE and discarding the TNE and blotting the cuvette and replacing a fresh aliquot of 2 ml TNE.
10) 2µl of sample was added and pipette-pumped in 2ml of TNE and the reading taken. Washings were carried out between samples, as before.
Notes.
The crystal-cuvette was always used in the same orientation and never handled on the sides exposed for fluorescent analysis.
The regression curve generated using the DNA standard allowed the calculation of sample DNA concentration from the fluorescence values.
--------------------------------------------------------------------------------
SILVER STAINING
The silver-staining method (Sambrook et al., 1989) has been adapted for a number of uses including detection of RFLPs, sequencing and microsatellites, but here we describe a quick protocol used to stain (4 - 8 %) polyacrylamide gels for RFLPs. The entire development procedure occurs under gentle agitation. The gels are soaked in buffer A (180 ml sterile H2O, 40 ml 100% EtOH, 2ml of glacial acetic acid) for four minutes. This buffer is drained and Buffer B (sterile H2O 100 ml, Silver nitrate 0.1 g) is added for 10 mins. Two 1 min washes in sterile H2O follow. Next the gel is soaked in Buffer C (sterile H2O 150 ml, NaOH 2.25g, NaBH4 (Sodium Borohydride) 15 mg, Formaldehyde 0.6 mls) until bands appear, but over exposure will darken the gel background. A 1 min rinse in sterile H2O is followed by fixing in buffer A again.
--------------------------------------------------------------------------------
BIOTIN LABELLING AND SIGNAL DETECTION SYSTEMS
Biotin is a vitamin that can be chemically-linked to purine or pyrimidine bases. In the form of a biotinylated nucleotide (e.g. bio-11-dUTP: the number denotes the length of the allylamine linker arm attaching the biotin molecule to the nucleotide), it can be incorporated in DNA probes using any of the methods described in Hybridisation Reactions. Biotin can also be incorporated into nucleic acids by linking it to aryl azide. Photobiotin produced in this manner reacts in visible light to produce aryl nitrene which binds to DNA. The biotin label that is incorporated in these numerous ways can now be detected using a variety of signal-generating systems (e.g. colloidal gold, fluorescein, 32S dATP). Biotin-labelled probes are first localised with antibodies, or more commonly, with avidin and streptavidin. Avidin and streptavidin are proteins with extremely high affinities for biotin; the second leads to a reduction of non-specific binding to DNA. It is to these moieties (biotin-steptavidin) that signal-generating systems attach to. For filter hybridisation, e.g. RFLPs, the enzymes used in conjunction with streptavidin are horseradish peroxidase (HRP) or alkaline phosphate. These produce visible products over a period of 4 h in the presence of suitable substrates, e.g. 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) (Mundy et al., 1991) and aryl phosphate-substituted 1,2-dioxetane in combination with a fluorescent enhancer system. In solid substrates such as microscope slides, cytochemical detection takes place using fluoresceins, e.g. fluorescein-avidin DCS. The intensity of the biotin-linked fluorescence is amplified by adding a layer of biotinylated goat anti-avidin antibody followed by another layer of fluorescein-avidin DCS. After washing off the excess stain, a fluorescence anti fade solution, e.g. p-phenylenediamine, is added in conjunction with a DNA counterstain 4,6-diamidino-2-phenylindole (DAPI) or propidium iodide that stain the chromosomal bodies blue (Pinkel et al., 1986). Note the fluorphores are available in a variety of colours - red, green, or blue 。
--------------------------------------------------------------------------------
AGAROSE-GEL METHOD WITH ETHIDIUM BROMIDE STAINING
1) The DNA for assessment was removed from cold storage and heated at 37oC for 30 min (or 3 min at 60oC ). Flicking the bottom of tubes ensured that all the DNA has been suitably resuspended.
2) The samples were then given a quick spin in a microcentrifuge and kept on ice prior to use.
3) 1µl of the DNA sample + 2µl loading dye + 10 µl sterile distilled water were mixed together.
4) Then additional tubes were set up comprising standard DNA (lambda or calf thymus DNA are routinely used in calibration. In general eukaryotic DNA is best used for eukaryotic samples.)
c. 25 ng/µl 0.5µl calf thymus DNA
(50ug/ml stock) + 2µl loading dye + 10.5µl sterile distilled water
c. 50 ng/µl 1.0µl " + " + 10.0µl "
c. 100 ng/µl 2.0µl " + " + 9.0µl "
c. 200 ng/µl 4.0µl " + " + 7.0µl "
5) These tubes were given a short spin.
6) The samples were loaded with the DNA standards at one end of a row of samples.
7) For a rough guide. Commercially available Lambda Hind III marker is used at 500 ng/ml conc. Heated at 60oC for 3 min, the marker (mixed with a suitable loading dye - see section on MOLECULAR TERMS AND SOLUTIONS) can indicate DNA concentration because its heaviest (20 kb band) approximates to 250 ng/µl of DNA concentration and the next two bands are 125 ng/µl and 67.5 ng/µl respectively.
7) The gel was typically run (at 100v) for 30 to 60 min.
8) The technique above was adapted for use with 6% polyacrylamide gels. 10µl volumes of the samples and standard DNA samples were added to the wells of a horizontal gel and the sample was run for 3h at 200v. After this the gel was stained using a silver stain procedure, described below.
--------------------------------------------------------------------------------
SPECTOROPHOTOMETER-ABSORBANCE ASSAYS
1) The DNA for assessment was removed from cold storage and heated at 37oC for 30 min (or 3 min at 60oC ). Flicking the bottom of tubes ensured that all the DNA has been suitably resuspended.
2) The samples were then given a quick spin in a microcentrifuge and kept on ice prior to use.
3) On older models of the spectrometers, the deuterium lamp was turned on 30 min before analysis to allow the lamp to heat up.
4) 2.00 ml of Filtered TE were aliquoted into 10mm dual light path disposable cuvette (Hughes & Hughes Ltd.) and used to zero the spectrometer = 1st cuvette.
5) In a second disposable cuvette 10µl of the DNA sample was pipette pumped in 1.99 ml of TE = sample cuvette. After zeroing the spectrometer on the 1st cuvette, the sample cuvette was brought into place to obtain an absorbance reading.
6) Readings were taken at wavelengths of 260 nm (OD260) and 280 nm (OD280) (the spectrometer was rezeroed between each wavelength reading).
(260 nm for nucleic acids and 280nm for protein concentrations).
The ratio between the readings (OD260/OD280) provided an estimation of sample purity: pure preparations had values close to 1.8 and protein contaminated samples were significantly lower. OD260 also allowed the calculation of nucleic acid concentrations of the samples, i.e. an OD of 1 corresponded to approximately c.50µg /ml for double stranded DNA.
--------------------------------------------------------------------------------
DNA FLUOROMETRY
1) The DNA for assessment was removed from cold storage and heated at 37oC for 30 min (or 3 min at 60oC ). Flicking the bottom of tubes ensured that all the DNA has been suitably resuspended.
2) The samples were then given a quick spin in a microcentrifuge and kept on ice prior to use.
3) On older models of the fluorometers, the machine was turned on 30 min before analysis or until the read-out had stabilised.
4) (For low concentrations of DNA, a eukaryotic 50 µl stock of 25ng/µl calf thymus DNA was used.)
5) Hoechst stain (1mg/ml) was diluted in 25 ml of 1 x TNE (filtered: 10mM Tris, 1mM EDTA, 0.1M NaCl) to a final concentration of 100ng/ml. The solutions were kept in 25 ml sterile specimen bottles (Granier) which were wrapped in silver foil to protect against light.
6) The fluorometer was zeroed using 2 ml of TNE.
7) The fluorometer was then first standardised using 100ng of DNA stock (25ng/µl) (8µl of standard in 2 mL of TNE). The solution was pipette-pumped using a 1000µl Gilson directly in the crystal cuvette and then the scale setting was adjusted to 100 for subsequent readings.
8) This was then followed by standards of 25 ng (2µl), 50ng (4µl), 75ng (6µl), 125ng (10µl) and 150ng (12 µl) of DNA stock in fresh 2 ml volumes of TNE.
9) Washes of the cuvette were carried out by tipping out the old sample and washing with a 1 ml aliquot of TNE and discarding the TNE and blotting the cuvette and replacing a fresh aliquot of 2 ml TNE.
10) 2µl of sample was added and pipette-pumped in 2ml of TNE and the reading taken. Washings were carried out between samples, as before.
Notes.
The crystal-cuvette was always used in the same orientation and never handled on the sides exposed for fluorescent analysis.
The regression curve generated using the DNA standard allowed the calculation of sample DNA concentration from the fluorescence values.
--------------------------------------------------------------------------------
SILVER STAINING
The silver-staining method (Sambrook et al., 1989) has been adapted for a number of uses including detection of RFLPs, sequencing and microsatellites, but here we describe a quick protocol used to stain (4 - 8 %) polyacrylamide gels for RFLPs. The entire development procedure occurs under gentle agitation. The gels are soaked in buffer A (180 ml sterile H2O, 40 ml 100% EtOH, 2ml of glacial acetic acid) for four minutes. This buffer is drained and Buffer B (sterile H2O 100 ml, Silver nitrate 0.1 g) is added for 10 mins. Two 1 min washes in sterile H2O follow. Next the gel is soaked in Buffer C (sterile H2O 150 ml, NaOH 2.25g, NaBH4 (Sodium Borohydride) 15 mg, Formaldehyde 0.6 mls) until bands appear, but over exposure will darken the gel background. A 1 min rinse in sterile H2O is followed by fixing in buffer A again.
--------------------------------------------------------------------------------
BIOTIN LABELLING AND SIGNAL DETECTION SYSTEMS
Biotin is a vitamin that can be chemically-linked to purine or pyrimidine bases. In the form of a biotinylated nucleotide (e.g. bio-11-dUTP: the number denotes the length of the allylamine linker arm attaching the biotin molecule to the nucleotide), it can be incorporated in DNA probes using any of the methods described in Hybridisation Reactions. Biotin can also be incorporated into nucleic acids by linking it to aryl azide. Photobiotin produced in this manner reacts in visible light to produce aryl nitrene which binds to DNA. The biotin label that is incorporated in these numerous ways can now be detected using a variety of signal-generating systems (e.g. colloidal gold, fluorescein, 32S dATP). Biotin-labelled probes are first localised with antibodies, or more commonly, with avidin and streptavidin. Avidin and streptavidin are proteins with extremely high affinities for biotin; the second leads to a reduction of non-specific binding to DNA. It is to these moieties (biotin-steptavidin) that signal-generating systems attach to. For filter hybridisation, e.g. RFLPs, the enzymes used in conjunction with streptavidin are horseradish peroxidase (HRP) or alkaline phosphate. These produce visible products over a period of 4 h in the presence of suitable substrates, e.g. 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) (Mundy et al., 1991) and aryl phosphate-substituted 1,2-dioxetane in combination with a fluorescent enhancer system. In solid substrates such as microscope slides, cytochemical detection takes place using fluoresceins, e.g. fluorescein-avidin DCS. The intensity of the biotin-linked fluorescence is amplified by adding a layer of biotinylated goat anti-avidin antibody followed by another layer of fluorescein-avidin DCS. After washing off the excess stain, a fluorescence anti fade solution, e.g. p-phenylenediamine, is added in conjunction with a DNA counterstain 4,6-diamidino-2-phenylindole (DAPI) or propidium iodide that stain the chromosomal bodies blue (Pinkel et al., 1986). Note the fluorphores are available in a variety of colours - red, green, or blue 。
