A total of 2ug of genomic DNA or cDNA is required for a single bi-directional ablation.Most methods for isolating RNA and genomic DNA are suitable for this experiment (Sambrooketal.1989;ChomczynskiandSacchi1987;Farrell1993). This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Experiments for the preparation of ablated cDNA or genomic DNA libraries
Materials and Instruments
Buffers, solutions and reagents Enzymes and enzyme buffers Nucleic acids and oligonucleotides Radioactive compounds Phenols Move Phase 1: Isolation of RNA and DNA For more product details, please visit Aladdin Scientific website.
Thermocyclers Water baths Reagents and equipment needed for agarose gel electrophoresis
A total of 2ug of genomic DNA or cDNA is required for a single bi-directional ablation.Most methods for isolating RNA and genomic DNA are applicable to this experiment (Sambrooketal.1989; Chomczynskiand Sacchi 1987; Farrell1993). If possible, the same reagents and protocols were used to purify the samples to be compared whenever possible. In the case of genomic DNA as the initial material, the next step is Rsa I digestion (stage 3 of this protocol); in the case of RNA as the initial material, the next step is cDNA synthesis (stage 2 of this protocol).
Stage 2: cDNA synthesis
I. Materials
1. Buffers, solutions and reagents
dNTP solution (10mmol/L)
DTT,0.1mol/L
EDTA,0.2mol/L
First chain buffer, 5X
Mineral oil
The SSH technique requires very precise PCR and in the authors' experience, mineral oil is necessary to maintain precision. Only when mineral oil is not available and the samples are easier to analyze, use the hot cap for large scale PCR screening. Therefore the author strongly recommends not to use hot caps until protocol 5.
Second Strand Buffer, 5X
TN buffer (10 mmol/L Tris-HCl, pH 7.6, 10 mmol/L NaCl)
2. Enzyme and enzyme buffer
AMV reverse transcriptase (20U/ul)
3. Nucleic acids and oligonucleotides
Primers for cDNA synthesis (10umol/L)
poly(A )+RNA
4.Radioactive compounds
Optional: [ a-32P ]dCTP (10mCi/ml, 3000Ci/mmol) (lCi=3.7X1010Bq)
5. Specialized equipment
Thermal cycler, or water baths preset to 16°C, 42°C and 72°C
6. Additional reagents
Phenol: reagents required for chloroform extraction and ethanol precipitation
II. METHODS
1. Synthesis of first strand cDNA
(1) In a 0.5 ml centrifuge tube, mix each test and drive sample with the following components.
poly(A)+RNA (2ug ) 2~4ul
cDNA synthesis primer (10umol/L) 1ul
H20 Add to 5ul
(2) Cover the sample surface with a drop of mineral oil and incubate for 2 min at 72°C in a thermal cycler or water bath.
(3) Cool at room temperature for 2min.
(4) Add the following ingredients to each reaction tube.
First chain buffer, 5X 2ul
dNTP solution (10 mmol/L each) 1ul
H2undefined 0.5ul
DTT,0.1mol/L 0.5ul
AMV reverse transcriptase (20U/u1) 1ul
~undefined optional: to detect the progress of cDNA synthesis, add 0.5ul [ a-32P ] dCTP (10mCi/ml,3000Ci/mmol) to 9ul H20, and take 0.5ul of diluted marker instead of the above H20
(5) Gently vortex to mix and centrifuge briefly.
(6) Warm the centrifuge tube at 42°C for 90 min in a thermal cycler or water bath.
(7) Place the centrifuge tube on ice, abort the synthesis of the first-strand cDNA, and immediately proceed to the synthesis of the second-strand cDNA.
2. Synthesis of second strand cDNA
(8) Add the following components (pre-cooled on ice) to the first strand synthesis tube.
Sterilized H2O 48.4ul
Second chain buffer, 5X 16.0ul
dNTP solution (10mmol/L) 1.6ul
Second strand synthase mixture, 20X 4.0ul
(9) Mix and centrifuge briefly, final volume should be 80ul.
(10) Warm the centrifuge tube at 16°C (water bath or thermal cycler) for 2h.
(11) Add 2ul (6U) of T4DNA polymerase and mix well.
(12) Warm the centrifuge tube at 16°C for another 30 min in a water bath or thermal cycler.
(13) Add 4ul 0.2mol/LEDTA,abort the synthesis of the second strand.
(14) Perform phenol:chloroform extraction and ethanol precipitation.
(15) Re-dissolve the precipitate with 50ul TN buffer.
(16) Take 6ul from the above solution, add to a new centrifuge tube and store at -20°C until after Rsa I digestion.
This sample will be subjected to agarose gel electrophoresis to estimate the yield and size range of dscDNA synthesis products.
Stage 3: Rsa I digestion
Perform the following procedure to process the double-stranded driver and assay DNA samples used for each experiment. This step produces short, flat-ended dsDNA fragments that are best suited for subtractive hybridization.
I. Materials
1. Buffers, solutions, and reagents
EDTA, 0.2mol/L
TN buffer (10 mmol/L Tris-HCl, pH 7.6, 10 mmol/L NaCl)
2. Enzyme and enzyme buffer
RsaⅠ(10U/ul)
Restriction buffer, 10X
3. Nucleic acids and oligonucleotides
Genomic DNA from phase 1, or cDNA from phase 2, 2ug per bi-directional subtraction reaction
4. specialized equipment
Water bath, preset at 37°C
5. Additional reagents
Phenol: reagent required for chloroform extraction and ethanol precipitation
Reagents and equipment required for agarose gel electrophoresis
II. Methods
1. add the following reagents to the centrifuge tubes or genomic DNA (gDNA) samples from Step 8 of Phase 2 [Protocol 2 - edited] above.
ds cDNA or genomic DNA 43.5ul
Ruling 1 Restriction Buffer, 10X 5.0ul
RsaⅠ(10U/ul) 1.5ul
2. mix well and incubate at 37°C for 2~4 h.
3. Determine the efficiency of RsaⅠdigestion by analyzing 5ul of digested product by 2% agarose gel electrophoresis, and use undigested DNA [genomic DNA from phase 1 (original text is Scheme 1 - edited by the editors)] as a control. Continue digestion during electrophoresis until you are satisfied with the results before discontinuing the reaction.
4. Add 2.5ul of 0.2mol/LEDTA and abort the digestion reaction. Extract with phenol: chloroform and collect DNA by ethanol precipitation.
The use of glycogen or any type of co-precipitant is not recommended for this step. These reagents may increase the viscosity of the DNA solution and inhibit subsequent DNA hybridization. Silica-based PCR purification systems should also be avoided.
5. Dissolve each precipitate in 6ul of TN buffer (no H20 ) and store at -20°C. This completes the preparation of the driver DNA.
Stage 4: Junctions
It is highly recommended that each pair of assay/driver DNA be ablated in both directions. Forward elimination is designed to enrich for differential molecules that are present in the detector but not in the driver, and reverse elimination is designed to enrich for differential molecules that are present in the driver but not in the detector. Utilizing both forward and reverse ablation of DNA is useful for the final differential screening of ablated detector DNA libraries (Scheme 6). Detection DNA is ligated to Adi (Detector 1-1 vs. 2-1) and Ad2R (Detector 1-2 vs. 2-2), respectively. A double junction (Adi and Ad2R) should also be made with the assay DNA (unabated assay controls 1-c and 2-c) as a negative control for the ablation reaction. The junctions do not connect to the driver DNA.
I. MATERIALS
1. Buffers, solutions and reagents
ATP, 3 mmol/L
EDTA,0.2mol/L
Mineral oil
TN buffer (10 mmol/L Tris-HCl, pH 7.6, 10 mmol/L NaCl)
2. Enzyme and enzyme buffer
Ligase buffer, 10X
T4 DNA Ligase (400 U/ul)
3. Nucleic acids and oligonucleotides
Adapter Ad1 (10umol/L)
Splice Ad2R (10umol/L)
Detection of RsaⅠdigestion DNA
4. Specialized equipment
Water bath, preset 37°C
5. Additional reagents
Phenol: reagents required for chloroform extraction and ethanol precipitation
Reagents and equipment required for agarose gel electrophoresis
II. Methods
1. Take out 1ul from the above RsaⅠdigested test DNA and dilute it with 5ul TN buffer.
2. Prepare a subject ligation mix for each reaction according to the following composition.
H2O 2ul
Ligation buffer, 5X 2ul
ATP(3 mmol/L) 1ul
T4 DNA Ligase (400 U/ul) 1ul
3. For each assay DNA mixture, add to a 0.5 ml centrifuge tube with the following reagents in the order shown. Pipet the solution to mix thoroughly. 
4. In a new centrifuge tube, mix 2^1 of Detector 1-1 with 24 of Detector 1-2. This serves as non-abated Detector Control 1-c. Do the same for each assayed DNA sample. After ligation, approximately 1/3 of the DNA molecules in each of the unabated detector control tubes will have been spliced to two different junctions, making them suitable for exponential PCR amplification using primers with junctions.
5. Cover the samples with a drop of mineral oil, centrifuge briefly, and incubate at 16°C overnight.
6. Add 1ul of 0.2mol/LEDTA to stop the ligation reaction.
7. Inactivate the ligase by treating the sample at 72°C for 5 min.
8. Remove 1ul from each unabated detector control (1-c, 2-c...) and dilute with 1ml TN buffer. This sample will be used for PCR amplification (Scheme 3).
The assay DNA1-1 and 1-2 for the ligated junctions in the experiment have been prepared up to this point.
9. Before moving on to the next part of the experiment, a ligation efficiency test is performed.
Phase 5: Ligation Efficiency Assay
I. Materials
1. buffers, solutions and reagents
dNTP solution (contains all 4 dNTPs, 10 mmol/L each)
Mineral oil
2. enzymes and enzyme buffers
PCR buffer, 10X (40 mmol/L Tricine-KOH, pH 9.2 at 22°C; 3.5 mmol/L magnesium acetate; 10 mmol/L potassium acetate; 75 mg/ml BSA, or vendor supplied)
Polymerase mix, 50X (Advantage2, Clontech, or equivalent)
3. Nucleic acids and oligonucleotides
PCR Primer P1
Unabated control sample (Step 4 of Phase 4 of Protocol 1)
4. Specialized equipment
Thermal cycler
5. Additional reagents
Reagents and equipment required for agarose gel electrophoresis
II. METHODS
1. 1ul of each unabated control sample was dispensed into 0.5 ml PCR tubes.
2. Mix the following reagents to prepare a master kunai for all reactions.
H2O 19.5ul
PCR buffer, 10X 2.5ul
dNTP solution (10 mmol/L per tube) 0.5ul
PCR Primer P1 1.0ul
Polymerase mix, 50X 0.5ul
Total Volume 24.0ul
3. Mix and centrifuge briefly.
4. Dispense 24ul of Master Mix into each reaction tube prepared in step 1.
5. Cover the sample with 1 drop of mineral oil.
6. Place the reaction mixture in a thermal cycler and incubate at 72°C for 5 min to extend the joints.
7. Immediately perform the following thermal cycle. 
8. Take 4ul of each tube and analyze on a 2% agarose gel.
Key: This PCR product should have a pattern similar to that of RsaⅠdigested DNA. If no PCR product is visible after 15 cycles, add 3 more cycles and analyze the product. If no product is visible after 21 cycles, analyze the product. If no PCR product is visible after 21 cycles, the activity of the polymerase mix should be checked. If there is no problem with the activity of the polymerase mix or other PCR reagents, the ligation reaction should be repeated with fresh samples before proceeding to the next step.