Genomic DNA can be isolated by a variety of methods, depending primarily on the type (blood or tissue) and amount of initial material. All prepared DNA should be stored in TE buffer pH 8.0 in sterilized Eppendorf tubes. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Preparation of template DNA, organization of experiments and PCR amplification experiments
Materials and Instruments
PCR buffer ddH20 Genomic DNA dNTP Oligonucleotide primers Move I. Materials For more product details, please visit Aladdin Scientific website.
Centrifuges and rotors Acrylic guards Filter-blocked tips Multichannel pipettes PCR instruments Trays Fixer devices Pedestal tube caps Small tubes
1. Buffers, solutions and reagents
10XPCR buffer containing 15 mmol/LMgCl2 (GeneAmpPCR Buffer I, AppliedBiosystems)
sterilized ddH20
2. Enzyme and enzyme buffer
TaqDNA polymerase, (AppliedBiosystems or Invitrogen)
3. nucleic acids and oligonucleotides
Genomic DNA
10 mmol/L dNTP (GeneAmpPCRdNTP mixture, AppliedBiosystems)
Oligonucleotide primers (synthesized by Invitrogen, diluted to lOumol/L)
4. Centrifuge and rotor
Centrifugation was performed with a tipping bucket rotor and microplate adapter.
5. Specialized equipment
acrylicshield
Filterbarriertips
Multichannel pipettes (Eppendorf or ApogentDiscoveries)
PCR instrument, 96-well (AppliedBiosystemsGeneAmp9700)
Tray/Fixer Unit and Base, 96-well (AppliedBiosystems)
Tube Caps, strip of 8 (USAScientific or AppliedBiosystems)
Tubes, 0.2 ml, 8 per strip (USAScientific or AppliedBiosystems)
Methods
1. melt dNTP, 10XPCR buffer, and unlabeled primers on ice. taq enzyme should be stored at -20°C prior to addition to the reaction mixture. melt the labeled primers behind an acrylic shield.
2. the mixture for each reaction consists of:
Sterilized ddH20 6.95ul
10X PCR buffer with MgCl2 1.25ul
10mmol/LdNTP 0.25ul
10mmol/L unlabeled primer 0.5ul
Labeled primer 0.5ul
During optimization of the experimental conditions, if the radiated signal intensity of the PCR product is too low, the amount of labeled primer can be increased, and the amount of labeled primer can be increased by the appropriate amount.
The volume of ddH20 should be adjusted accordingly.
TaqDNA polymerase (final concentration 0.25 U) 0.05ul
For a 96-well microplate, prepare a mixture for 100 reactions.
3. Mix well and add in aliquots to a PCR plate placed on ice (9.5ul/sample).
4. Add 3ul of DNA (60ng) using a multichannel pipette and mix well.
5. Put the lid on tightly and place the plate in the PCR instrument preheated to 94°C.
6. If the product size is 200bp, amplify 30 cycles. The cycle parameters are as follows: initial denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30s; annealing at an empirically determined annealing temperature (Tm) for 30s; extension at 72°C for 30s; and a final step of extension at 72°C for 7 min. Values can be calculated from the primer sequences by using the formula 2(A+T)+4(G+C).
7. After amplification is complete, freeze the samples at -20°C or go directly to Protocol 3.