Hybridization experiments with oligonucleotide probes in solution
Hybridization experiments with oligonucleotide probes in solution
Jabobs et al. (1988) described the method and principles of hybridization in quaternary ammonium-containing buffers. The following is a simple variation of this method. This experiment was obtained from Molecular Cloning Laboratory Guide (Third Edition), Book 1, by Peitang Huang.
Operation method
Hybridization experiments with oligonucleotide probes in solution
Materials and Instruments
Oligonucleotide Hybridization Solution Oligonucleotide Pre-Hybridization Solution SSC or SSPE TEAC1 Wash TMAC1 or TEACl TMAC1 Wash Nucleic Acids and Oligonucleotides Radiolabeled Oligonucleotide Probes Move makings For more product details, please visit Aladdin Scientific website.
Hybridization device Vibratory incubator
Solutions and buffers
Dilute the storage solution to the appropriate concentration.
Oligonucleotide Hybridization Solution
6XSSC (or 6XSSPE)
0.05mol/L sodium phosphate (pH 6.8)
1 mmol/L LEDTA (pH 8.0)
5X Denhardt's liquid
100ug/ml denatured, broken salmon sperm DNA (Pharmacia Corporation or prepared as described in Scheme 10 of Chapter 6)
100 mg/ml Dextrose sulfate (see DEAE-Dextrose in the information section of Chapter 16)
Oligonucleotide prehybridization solution
6XSSC (or 6XSSPE)
0.05mol/L sodium phosphate (pH 6.8)
1 mmol/L EDTA (pH 8.0)
5X Denhandt's solution
100ug/ml denatured, broken salmon sperm DNA (Pharmacia or prepared as described in Scheme 10 of Chapter 6)
6xSSC or SSPE
Place this solution on ice before steps 4 and 5 of this protocol.
TEAC1 Wash Solution
2.4mol/LTEACl
50 mmol/LTris-Cl (pH 8.0)
0.2 mmol/LEDTA (pH 7.6)
1 mg/ml SDS
This wash solution is used for probes of 50 to 200 nucleotides in length. It must be pre-warmed to the desired temperature before use (see steps 6 and 7).
TMAC1 (5mol/L) or TEACl (3mol/L)
Prepare an aqueous solution of 5mol/LTMAC1 or 3mol/LTEAC1. TMACl is used for probes from 14 to 50 nucleotides in length, while TEACl is used for probes from 50 to 200 nucleotides in length. Both reagents are available from Aldrih. To the TMAC1 and TEAC1 solutions, activated carbon is added at a final concentration of approximately 10% and allowed to fall for 20-30 minutes. After the activated carbon has settled, the quaternary ammonium solutions are filtered through Whatman No. 1 paper and filtered through a nitrocellulose membrane (0.45um pore size) for sterilization. The refractive index of the solution is measured and the concentration of the quaternary ammonium salt solution is accurately calculated using the following formula: C=(n-1.331)/0.018, where C is the molar concentration of the quaternary ammonium salt solution and n is the refractive index. The filtered solution can be stored in a brown bottle at room temperature.
TMAC1 Wash Solution
3.0 mol/LTMAC1
50 mmol/LTris-Cl (pH 8.0)
0.2 mmol/LEDTA (pH 7.6)
1 mg/ml SDS
This wash solution is intended for use with probes 14-50 nucleotides in length and must be pre-warmed to the desired temperature prior to use (see steps 6 and 7).
Nucleic Acids and Oligonucleotides
Target nucleic acids immobilized on nitrocellulose, nylon or other filter membranes (Southern or Norths blots, lysed bacterial clones into phage-phagocytosed membranes)
Probes
Radiolabeled oligonucleotide probes
The probes are prepared as described in Scheme 2 or Scheme 7. We recommend the use of phosphorylated probes, which are purified by CPB precipitation as described in Scheme 4 prior to hybridization. This purification method removes unadulterated [ γ-32P ]ATP, thereby reducing the number of strongly radioactive spots (pepper spots) that can be easily confused with positive hybridization signals during radioactive autoradiography.
Specialized equipment
Hybridization device (see step 1)
Pre-warm the incubator, water bath or hybridization device to 37°C (steps 1 and 3) and adjust to the appropriate wash temperature during washing (step 7).
Methods
1. Prehybridize filters or membranes in oligonucleotide prehybridization solution at 37°C for 4 to 16 h.
Pre-hybridization, hybridization and washing of circular membranes are best performed in Sears Seal-A-Meal bags or plastic cases with sealed lids, while Southern and Northern hybridization membranes can be performed in glass tubes with sealed lids.
2. Remove the prehybridization solution and replace with a hybridization solution containing 180 ppmol/L radiolabeled oligonucleotide probes.
When several oligonucleotides are hybridized simultaneously, the concentration of each probe should be 180 pmol/L and the specific activity of the radiolabeled probe should be between 5X105 and 1. 5X106 cpm/pmol.
3. The membrane should be incubated at 37°C for 12 to 16 hours.
4. Dispose of the radioactive hybridization solution into a suitable container and wash the membrane three times at 4°C with pre-cooled 6XSSC or 6XSSPE to remove most of the dextran sulfate.
5. Wash the membrane twice with pre-cooled 6XSSC or 6XSSPE at 4°C for 30 minutes.
6. Wash the membrane twice at 37°C with TMAC1 or TEAC1 solution.
The purpose of this step is to replace the SSC or SSPE with a quaternary ammonium solution, which is superior only if done carefully.
7. Wash the membrane twice with TMAC1 or TEAC1 Wash Solution for 20 minutes at temperatures 2 to 4°C below the Tm values indicated in Figure 10-2.
The Tm value of hybrids in TEAC1-containing buffer is 33°C lower than in TMACl-containing buffer. Make sure that the buffer is pre-warmed to the desired temperature and that the temperature fluctuation is less than ± 1°C.
8. Remove the membrane from the wash solution, aspirate the liquid at room temperature and perform radioautography as described in Appendix 9.