Differential hybridization experiments with detection and drive DNA probes
Differential hybridization experiments with detection and drive DNA probes
The following 4 probes are used for differential screening hybridization.
1. detector-specific ablation probe (forward ablation probe). 2. driver-specific ablation probe (reverse ablation probe).
2. kinesiology-specific ablation probes (reverse ablation probes).
3. cDNA probes synthesized directly from detector mRNA (or detector genomic DNA). 4.
4. cDNA probe synthesized directly from driver mRNA (or driver genomic DNA).
This experiment is derived from PCR Laboratory Guide (2nd edition) by Seed Kang and Qu Lijia.
Operation method
Differential hybridization experiments with detection and drive DNA probes
Materials and Instruments
Wash buffer SDS SSC Blocking solution Sheared salmon sperm DNA probe Move I. Materials For more product details, please visit Aladdin Scientific website.
Boiling water bath Hybridization oven
1. Buffers, solutions and reagents
High stringency wash buffer (0.2XSSC, 5 g/L SDS), preheated to 68°C (optional, see step 9)
Low stringency wash buffer (2XSSC, 5 g/LSDS), preheated to 68°C (optional, see step 9)
SDS, 5 g/L (optional, see step 9)
SSC, 20X (1L formulation: 175.3 g NaCl and 88.2 g sodium citrate, pH 7.0)
2. Nucleic acids and oligonucleotides
Blocking solution [contains 2 mg/ml of unpurified NP1, NP2R, cDNA synthesis primers (in case of cDNA), and their complementary oligonucleotides].
Sheared salmon sperm DNA (10 mg/ml)
3. probes
Purified detector and driver probes, labeled by random priming (at least 107 cpm per 100ng of ablated DNA) (optional, see step 9)
4. Specialized equipment
Boiling water bath
Hybridization oven, preheated to 72°C
5. Additional reagents
ExpressHyb Hybridization Kit (Clontech)
Reagents and equipment required for radiographic autoradiography
II. Methods
1. The DNA probes were labeled for drive and detection by the random primer method using commercial kits. The hybridization conditions given here are optimized for Cbntech's ExpressHyb solution; for other systems, optimal hybridization conditions should be determined empirically.
2. Prepare a sufficient volume of pre-hybridization solution for each membrane.
a. Mix the following solutions: 5ul of 20XSSC, 5ul of sheared salmon sperm DNA (10 mg/ml), and 10ul of blocking solution [containing 2 mg/ml of unpurified NP1, NP2R, cDNA synthetic primer (in case cDNA is used), and their complementary oligonucleotides]. b. Prepare the solution for each membrane. c. Prepare the solution for each membrane. d. Prepare the solution for each membrane.
b. Boil the blocking solution for 5 min, then cool on ice.
c. Mix the blocking solution with 5 ml of ExpressHyb Hybridization Solution (Clontech).
3. Place each flag in the prehybridization solution prepared in step 2 and prehybridize for 40-60 min at 72°C with oscillation.
It is important to add a blocking solution to the prehybridization solution because the subtraction probes have the same junction sequence as the clones being analyzed, and these probes will hybridize with all clones regardless of their internal sequence.
4. Preparation of hybridization probes.
a. Mix the following solutions: 50ul of 20XSSC, 50ul of sheared salmon sperm DNA (10 mg/ml), 10ul of blocking solution, and the purified probes (at least 107 cpm per 100ng of ablated DNA). Make sure that the specific activity of each probe is approximately equal.
b. Boil the probes for 5 min, then cool on ice.
c. Add the probes to the prehybridization solution.
5. Vibrate the hybridization at 72°C overnight.
6. Prepare low stringency (2XSSC,5 g/LSDS) and high stringency (0.2XSSC,5 g/LSDS) Wash Buffer and heat to 68°C.
7. Wash the membrane sequentially with Low Stringency Buffer (4X20 min, 68°C) and High Stringency Buffer (2X20 min, 68°C).
8. Perform radioactive autoradiography.
9. If desired, boil in 5 g/L SDS for 7 min to remove the probe.
The blot can generally be reused at least 5 times.
To minimize hybridization background, flags should be stored in SaranWrap at -20°C when not in use.