Northern blot analysis assay
Northern blot analysis assay
To confirm that the selected cDNAs are indeed differentially expressed, it is recommended that Northern blot analysis be used instead of other confirmatory techniques, such as reverse Northern hybridization (Zhangetal.1996) or quantitative RT-PCR.This experiment was derived from PCR Laboratory Guidelines (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Northern blot analysis assay
Materials and Instruments
Agarose gel Colony lysis buffer Distilled water Formaldehyde Formamide Pre-hybridization Hybridization solution HotPrime cDNA Labeling Kit Mineral oil MOPS Buffer MOPS Sodium acetate EDTA PCR buffer SSC NaCl SDS NaCl NaH2PO4 [a-32P]dATP Lgh Rgh Primer Salmon extract DNA Taq DNA Polymerase Move I. Materials For more product details, please visit Aladdin Scientific website.
Centrifuges Filter paper sheets Light intensifying screens Centrifuge tubes Cellulose nitrate or nylon membranes Pipettes Blink counters Sephadex G50 columns Single-sensor scientific imaging film Thermocyclers Thin-walled PCR tubes Plastic cling film
1. Buffers, solutions and reagents
1.5% agarose gel with ethidium bromide
Colony lysis buffer (PCR-TRAP Cloning System, GenHunter P404 or L102)
Denhardt solution (500 ml)
Ficoll 5 g
Polyvinylpyrrolidone (PVP) 5 g
BSA (PentaxFractionV) 5 g
Distilled water Add to 500 ml
Distilled water
12.3mol/L (37%) formaldehyde, pH>4.0
Formamide prehybridization/hybridization solution (GenHimterMLl), if preparing your own, use the following protocol (500 ml)
20XSSPE 125 ml
50XDenhardt solution 50 ml
20% SDS 2.5 ml
Formamide 250 ml
Evaporated water Add to 500 ml
Mix well, divide into smaller volumes and store at -20°C.
HotPrime cDNA Labeling Kit (GenHunterH50), including KlenowDNA Polymerase (1 U/ul), 10X Labeling Buffer, dNTP(-dATP) or dNTP(-dCTP) (500 umol/L), abort buffer, and distilled water.
Mineral oil (optional)
10XMOPS buffer
0.2mol/LMOPS
0.05mol/L sodium acetate
0.01mol/LEDTA
10XPCR buffer (RNAspectra Differential Display System, F501-F510&R501-R510,PCR-TRAP Cloning System, GenHunter, P404 or S201)
20XSSC
3mol/L NaCl
0.3mol/L trisodium citrate-2 H20
Adjust pH to 7.0 with 1mol/LHC1
1XSSC,1g/L SDS
0.25XSSC,1g/L SDS
20XSSPE
3mol/L NaCl
0. 1mol/LNaH2PO4 (second generation salt)
0.02mol/LEDTA
Abort buffer (0.lmol/LEDTA, pH 8.0)
2. Nucleic acids and oligonucleotides
[ a-32P ]dATP (3000Ci/umol/L) (1Ci=3.7X1010Bq)
dNTP (250umol/L) (PCR-TRAP Cloning System, GenHunter P404 or S501)
Lgh/Rgh primers (2umol/L) (PCR-TRAP Cloning System, GenHunter P404 or L201&L202)
Salmonid sperm DNA (GenHunter ML2)
3. Enzyme and enzyme buffer
Taq DNA polymerase (Qiagen201207)
4. Specialized equipment
Centrifuge
3 mm filter paper sheets
Light Enhancement Screen
1.5 ml tube
Nitrocellulose or nylon membrane
Pipette
Scintillation counter
Sephadex G50 column (Roche Applied Science 1814419, Indianapolis, IN)
Single-sensor scientific imaging film [Recommended Kodak Biomax MS, No. 8715187 (Kodak-Eastman, Rochester, NY)
Thermal cycler
0.2 ml thin-walled PCR tubes
UV-transparent plastic cling wrap [Recommended Glad Cling Wrap (The Glad Products Company, Oakland, CA)
5. Additional Reagents
QIAEX Ⅱ Gel Extraction Kit (Qiagen20021)
II. Methods
1. cDNA probe generation
(1) At the bottom of each cloning plate, number each tetracycline-resistant colony to be analyzed (at least 5 per plate is recommended).
(2) Dispense 5ul of Colony Lysis Buffer into each 1.5 ml centrifuge tube.
(3) Pick each colony with a clean pipette tip, trying not to take too much (a very small amount visible to the naked eye is usually enough). Transfer the cells to a correspondingly labeled 1.5 ml centrifuge tube.
(4) Warm the tubes in a boiling water bath for 10 min.
(5) Centrifuge at maximum speed for 2 min at room temperature to precipitate cellular debris. Transfer the supernatant to a clean 1.5 ml centrifuge tube and discard the residual precipitate.
(6) The lysate is used immediately for PCR analysis or stored at -20°C for backup.
(7) Add the following components to a 0.2 ml thin-walled PCR tube for colony PCR (it is highly recommended to prepare a core mix containing all components except the colony lysate to minimize errors in sample aspiration).
Distilled water 10.2ul
10XPCR buffer 2.0ul
dNTP mix (250umol/L) 1.6ul
Lgh primer 2.0ul
Rgh primer 2.0ul
Colony lysate 2.0ul
Taq DNA polymerase 0.2ul
Mix well and add 30ul of mineral oil if needed.
(8) Set up the thermal cycler according to the following program: 94°C 30s → 52°C 40s → 72°C lmin → 30 cycles → 72°C 5 min (extension) → 4°C hold.
(9) Run electrophoresis of all 20 ul of PCR products on a 1.5% agarose gel containing ethidium bromide dye. This not only generates the cDNA probe for Northern blot analysis, but also shows which of the cDNA clones generated using the PCR-TRAP CloningSystem (see Scheme 6) contains the correct insert.
(10) Purify each band from an agarose gel using the QIAEX II Gel Extraction Kit. The purified fragments are used as templates to generate probes with the HotPrime DNA Labeling Kit.
(11) If different vectors are used for cloning, generate cDNA probes for Northern blot hybridization according to the manufacturer's recommendations.
2. Labeling of cDNA probes
(12) If using the recommended HotPrime DNA Labeling Kit, place all components on ice immediately after complete thawing.
(13) Prepare the following reaction system in a 1.5 ml centrifuge tube with a fixed cap (so that the cap does not come loose during boiling).
Distilled water 11ul
10X labeling buffer 3ul
DNA template for labeling (10~50ng) 7ul
(14) Warm the reaction mixture in a boiling water bath for 10 min.
(15) Cool the reaction tube rapidly on ice. Centrifuge briefly to collect condensate.
(16) Add the reaction reagents in the order given below.
dNTP(-dATP)(500umol/L~undefined 3ul
[a-32P]dATP(3000Ci/mmol/L~undefined 5ul
Klenow DNA Polymerase 1ul
~undefined If [a-32P]dCTP is used instead of [a-32P]dATP, replace dNTP(-dATP) with dNTP(-dCTP).
(17) Allow the mixture to stand at room temperature for 20 min, then incubate at 37°C for 10 min.
(18) Add suspension buffer and mix well.
(19) Purify the labeled probe using a Sephadex G50 column. Collect the purified probe in a 1.5 ml centrifuge tube with a fixed cap. Count 1ul of labeled probe in a scintillation counter. For most labeled DNA probes, a total of 10 million or more cpm can be obtained.
3. Probe hybridization
(20) Protocols for the preparation of denaturing agarose (RNA) gels (including spiking and electrophoresis conditions, as well as transfer of RNA to nitrocellulose or nylon membranes) are described in Ausubel et al.
(21) If the prehybridization buffer is stored at -20°C, thaw at 37°C for 20 min.
(22) Denature salmon sperm DNA by warming in a boiling water bath for 10 min.
(23) Add salmonid DNA (final concentration 100~200ug/ml) to the prehybridization solution and mix well.
(24) Cover the membrane with 5 ml or a sufficient amount of prehybridization solution and prehybridize at 42°C for at least 4 hours.
(25) Using a 1.5 ml centrifuge tube with a fixed cap (otherwise the cap will come loose), boil the purified probe in a water bath for 10 min to denature the probe.
(26) Cool on ice for 2 min.
(27) Centrifuge to remove condensate and add the probe directly to the prehybridization solution.
(28) Hybridize overnight.
(29) Carefully pour out the radioactive hybridization solution and dispose of it in a special container for radioactive waste.
(30) Wash twice with 1XSSC containing 1 g/L SDS at room temperature. Handle the washing solution in a special container for both.
(31) Rinse with preheated 0.25XSSC containing 1g/L SDS at 50~55°C for 15~20 min.
4. Blot exposure
(32) Blot the membrane with 3 mm filter paper and wrap it with UV-permeable plastic film.
(33) Expose the blot to a monochromatic film with an intensifying screen for optimum detection signal at -70°C. The blot is then exposed to a UV-permeable plastic film.