Mirror direction selection experiment
Mirror direction selection experiment
The major disadvantage of SSH is the presence of background clones representing non-differentially expressed DNA species in the ablated library. In some cases, the number of background clones may greatly exceed the number of destination clones. To overcome this problem, mirror orientation selection (MOS) is recommended. This experiment was derived from PCR Lab Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Mirror direction selection experiment
Materials and Instruments
EDTA Mineral oil TN buffer PCR buffer Polymerase mix Xma Ⅰ Xma Ⅰ buffer DNAdNTP solution PCR primers for each dilution of the second hybridization sample Move Phase 1: PCR amplification for MOS and XmaⅠ digestion For more product details, please visit Aladdin Scientific website.
Thermal cycler Water bath Reagents and equipment required for agarose gel electrophoresis Phenol
I. Materials
1. Buffers, solutions and reagents
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
PCR buffer, 10X (40 mmol/LTricine-KOH, pH9.2 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)
Xma I (10U/ul)
Xma I buffer, 10X
3. Nucleic acids and oligonucleotides
DNA
dNTP solution (contains all 4 dNTPs, 10 mmol/L each)
Dilutions of second hybridization samples (from step 17 of Scheme 2)
PCR Primer P1
PCR primer NP2R
PCR Primer P1
4. Specialized equipment
Thermal cycler
Water bath, preset at 37°C and 74°C
5. Additional reagents
Reagents and equipment required for agarose gel electrophoresis
Phenol: reagents required for chloroform extraction and ethanol precipitation
II. METHODS
1. Primary PCR amplification for MOS
(1) Take 10ul of each dilution of the second hybridization product (from Step 17 of Scheme 2) and add to a labeled tube.
(2) Prepare the body mix for primary PCR-1 according to the table below. This recipe is sufficient for one reaction. Scale up as necessary. For each reaction, mix the reagents according to the table below. 
(3) Mix well and centrifuge briefly.
(4) Dispense 115ul of the master mix into the reaction tubes from step 1.
(5) Dispense the final 125ul of mixture into 5 0.5 ml (ul - translator's change) PCR tubes (25ul per tube).
(6) Cover with 1 drop of mineral oil.
(7) Incubate the reaction mixture in a thermal cycler at 74°C for 5 min to extend the joints, and then immediately start the following cycle. 
(8) From each of the 5 primary PCR-1 products, mix 2ul in a tube and add 390ul of H20.
(9) From each of the primary PCR-1 product mixes diluted in step 8, add 1ul each to a labeled PC tube.
(10) Prepare a master mix for primary PCR-2 according to the table below. 
(11) Mix well and centrifuge briefly.
(12) Dispense 24ul of Body Mix into each of the reaction tubes from step 9.
(13) Cover with 1 drop of mineral oil.
(14) Start the following cycle immediately. 
(15) From each reaction product, take 4ul of each and analyze on a 2.0% agarose gel.
2. Secondary PCR for MOS
(16) From each of the primary PCR-2 mixes generated in step 14 above, take 2ul of each and dilute with 38ulH20.
(17) Dispense 2ul of each diluted primary PCR-2 product mixture into labeled tubes.
(18) Prepare the master mix for secondary PCR according to the table below. This recipe is sufficient for one reaction and can be scaled up as needed. For each reaction, mix the reagents according to the table below. 
(19) Mix well and centrifuge briefly.
(20) Dispense 48ul of the master mix into the reaction tubes from step 2.
(21) Cover with 1 drop of mineral oil.
(22) Start 10-12 cycles immediately: 95°C 10s, 68°C 10s, 72°C 1.5 mm.
(23) Take 4ul of each reaction product and analyze on a 2.0% agarose gel.
(24) The secondary PCR products were purified by phenol/chloroform extraction and ethanol precipitation.
(25) Dissolve the precipitate with 20-40ul of TN buffer to achieve a DNA concentration of 20ng/ul.
(26) 2ul of the purified PCR product was analyzed on a 2.0% agarose/ethidium bromide gel.
(27) Dilute 14 the PCR product purified in step 10 with 1.6 ml H20 (this will serve as an undigested control).
(28) Store the reaction products at -20°C.
3. XmaI digestion
(29) Add the following reagents to the tube 
(30) Mix well and incubate for 2 h at 37°C.
(31) Add 2ul of 0.2mol/LEDTA to terminate the reaction.
(32) Incubate at 74°C for 5 min to inactivate the enzyme.
(33) Store at -20°C.
Phase 2: MOS hybridization
I. Materials
1. Buffers, solutions and reagents
Dilution buffer (20 mmol/LHEPES-HC1, pH 8.3,50 mmol/LNaCl and 0.2 mmol/LEDTA)
4X hybridization buffer storage solution: 4 mol/L NaCl, 200 mmol/L HEPES, pH 8.3, 4 mmol/L hexadecane
Triethylammonium Bromide (CTAB), final 1X hybridization mix with dilution buffer to dilute mineral oils
2. Nucleic acids and oligonucleotides
XmaI digested DNA
3. Specialized equipment
Thermal cycler
II. Methods
1. In a 1.5 ml centrifuge tube, mix the following reagents. 
2. From the mixture, dispense 2ul into a 0.5 ml centrifuge tube and cover with 1 drop of mineral oil.
3. Incubate in a thermal cycler at 98°C for 1.5 min.
4. incubate at 68°C for 3 h in a thermal cycler.
5. Add 200ul of Dilution Buffer to the tube and mix by pipetting.
6. Heat at 70°C for 7 min in a thermal cycler.
7. Store at -20°C.
Phase 3: MOSPCR amplification
I. Materials
1. buffers, solutions and reagents
dNTP solution (contains all 4 dNTPs, lOmmol/L each)
Mineral oil
2. enzymes and enzyme buffers
PCR buffer, lOX (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 as supplied by the manufacturer)
Polymerase Mix, 50X (Advantage2, Clontech, or equivalent)
3. Nucleic acids and oligonucleotides
Diluted DNA samples (hybridized samples and corresponding undigested controls, Step 6 of Protocol 4 Phase 2 and Step 26 of Protocol 4 Phase 1)
MOSPCR Primers (NP2Rs)
4. Specialized equipment
Thermal cycler
5. Additional reagents
Reagents and equipment required for agarose gel electrophoresis
II. METHODS
1. Prepare a body mix for all MOSPCRs according to the table below. 
2. Add lul of diluted DNA samples (post-hybridization samples and corresponding undigested controls) to each of the labeled tubes containing 24ul of body mix.
3. Cover with 1 drop of mineral oil.
4. Place the reaction mixture in a thermal cycler and incubate at 74°C for 5 min to extend the junction (do not remove the sample from the thermal cycler).
5. Start the following cycle immediately.
6. From each tube, take 4ul each to analyze on an agarose gel.
7. Store the reaction products at -20°C.