Primer design and end-labeling experiments
Primer design and end-labeling experiments
Systematic analysis of SSCP parsing capabilities and limitations revealed a significant correlation between sensitivity and fragment length. The highest detection rate of mutations was observed when the DNA length was 150-200bp. This protocol applies highly specific radioisotopes and includes a purification step for removal of unused nucleotides, thus reducing the radiation energy of the overall reaction. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Primer design and end-labeling experiments
Materials and Instruments
Kinase Buffer MgCl2 Dithiothreitol Bovine Serum Albumin ddH20 TE Buffer EDTA Polynucleotide Kinase Primer Radioactive Compounds Move I. Materials For more product details, please visit Aladdin Scientific website.
Radioactive Compound Centrifuges and Rotors Acrylic Sheeting Centrifuge Tube Racks Waste Containers Geiger Counter QuickSpin Columns
1. Buffers, solutions and reagents
5X kinase buffer
0.25mol/L Tris-HCl,pH9
0. 05mol/LMgCl2
0.05mol/L Dithiothreitol (DTT)
0.25 mg/ml bovine serum albumin (BSA)
-20°C Storage
Sterilized redistilled water ( ddH20 )
TE buffer, pH 8.0
10 mmol/L Tris-HCl, pH 8.0
1 mmol/LTris-HCl, pH 8.0
2. Enzyme and enzyme buffer
Polynucleotide kinase, 10 U/ul (Roche)
3. nucleic acids and oligonucleotides
Oligonucleotide primers
4. Radioactive compounds
[ y-32P ]ATP6000Ci/mmol/L (lCi=3. 7X1010Bq ) (PerkinElmerLifeSciences)
Also available is [ y-33P ]ATP, which is slightly less dangerous than [ y-32P ]ATP and has a longer half-life, but is more expensive.
5. Centrifuges and Rotors
Centrifugation with a tipping bucket rotor, adapter for 15 ml conical tubes
6. Specialized equipment
Acrylic shield, centrifuge tube rack ("betablock") and waste container
Centrifuge tube racks (e.g. 3008-1000fromUSAScientific)
Collection tubes (1.5 ml, including rapid centrifuge columns)
Coolingblock
Conical tube, 15 ml
Geigercounter (a radiometric energy meter)
Centrifuge tube, 0.5 ml, sterilized
QuickSpin column (TE:), G-25 dextran gel (Sephadex) column for purification of radiolabeled DNA (Roche)
Water bath
II.
1. Dilute the primers to 10 umol/L with sterilized ddH20. The purchased primers were synthesized at the 50 nmol/L level and received as a lyophilized powder. Dissolve the dry powder in TE buffer at pH 8.0 and store as a storage solution. Dilute to a 10umol/L working solution before use, dispense in small portions, and freeze at -20 °C.
2. Select a primer for end labeling. Thaw the primer and 5X kinase buffer on ice. Keep the enzyme in the freezer until it is added to the reactants, or use a benchtop cooling block to maintain the enzyme at -20°C. To melt the isotope at room temperature, hold the acrylic shield in front of you.
3. Add 3ul of sterilized ddH20, 5.6ul of 5X kinase buffer, 12ul of 10mol/L primer, and 1.4ul of polyglycolate kinase to a sterilized 0.5 ml centrifuge tube on ice, place the ice box behind the acrylic shield, and add 6ul of [ y-32P ] ATP (6000Ci/mmol/L). Mix well, cover and incubate for 1~2 h on a 37°C water bath.
4. Prepare the QuickSpin column during the last 10 min of incubation. Mix the column by inverting it repeatedly, remove the cap and stopper and place it in a collection tube. Place the column/collection tube assembly in a 15 ml conical tube and centrifuge in a benchtop centrifuge at 1100 g for 2 min. Invert the collection tube and centrifuge again. Transfer the column to another clean collection tube before loading the end-labeled reaction mixture.
5. After warming is complete, centrifuge the end-labeled reaction mixture briefly in a centrifuge. Add 52ul of sterilized ddH20, mix by pipetting, and transfer all solutions to the centrifuge column for purification (e.g., to remove unused nucleotides).
Centrifuge at 6.1100 g for 4 min.
7. Remove the column from the collection tube and discard into an appropriate radioactive waste container. Cap the collection tube in which the end marker was collected. Continue with the PCR protocol (Protocol 2) or freeze the primers at -20°C in a tube rack.