1. Genomic DNA isolation from whole blood or cells
To facilitate rapid genomic DNA isolation from fresh whole blood or frozen cell samples from up to 32 animals simultaneously, we use a MagNA Pure robot and MagNA Pure LC DNA isolation kit according to instructions provided by Roche Applied Science (https://www.roche-applied-science.com). Other DNA isolation techniques such as QIAamp DNA Blood Mini kits or DNeasy Blood and Tissue Kits from Qiagen (http://www.qiagen.com) can be substituted if this robot is not available. Regardless of isolation technique, proper protective equipment should be worn when handling nonhuman primate samples and DNA isolation reagents. Typical DNA concentrations from 300ul blood samples with the MagNA Pure LC DNA Isolation Kit range between 10–35 ng/ulin an elution volume of 100 ul.
(a) Ensure that the MagNA Pure LC Instrument has the proper DNA purification protocols installed as detailed by the MagNA Pure LC DNA Isolation Kit manual.
(b) Prepare the isolation kit reagents as detailed in the manual (https://www.roche-applied-science.com).
(c) Aliquot 300 ul of whole blood or white blood cells, or ~1 x 106 peripheral blood mononuclear or cultured cells/100 ul as directed by the manual.
(d) Set up the instrument using the appropriate protocol for the starting material. Instructions supplied by the software will calculate individual reagent volumes based upon number of samples to be isolated.
(e) Upon completion of the isolation run, quantify DNA concentrations with a NanoDropTM (Thermo Scientific) normalize DNA samples to 15 ng/ul prior to proceeding with PCR-SSP amplification. If necessary, samples can be stored indefinitely at -80 °C prior to PCR.
2. PCR-SSP amplification of Indian rhesus macaque MHC class I genomic DNA
[MJ Research Tetrad Thermocycler (Bio-Rad Laboratories), Thermal Activated Platinum Taq DNA Polymerase (100 μL, 5 units/μL) Invitrogen #10966034, Sterile Nuclease (DNase, RNase)-Free Water (Fisher Scientific #NC9628830) alternatively other thermocyclers may be used]
(a) Aliquot 12 μL of appropriate primers into strip-cap PCR tubes (see Indian rhesus PCR-SSP primers under Primer Information tab).
(b) Aliquot total volume of PCR mix (components and recipes below) needed for the experiment into a 1.5 mL tube containing the PCR mix (8 μL PCR mix per PCR reaction).
| PCR Mix | 5X PCR Buffer | dNTPs |
| 9280 μL PCR buffer |
300 mM Tris HCl | Promega dNTP set, 40 μmol each (catolog #U1240) |
| 2000 μL glycerol |
75 mM ammonium sulfate | Mix contents of 4 vials, dispense 320 μL aliquots into 1.5 mL tubes |
| 320 μL dNTPs |
10 mM MgCl2 | Store at -20°C until use |
| 360 μL ddH20 |
H2O to 500 mL | |
| 40 μL cresol red |
pH 9.5 | |
| store mix at 4°C up to 4 wks | Store 8 mL buffer + 1280 μL H2O in -20°C freezer until use |
(c) Add 0.6 uL Platinum Taq for each 40 μL PCR mix to the 1.5 mL tube containing the PCR mix. DO NOT VORTEX but pipet gently up and down to mix.
(d) Add the Taq/PCR mix to the diluted DNA and mix each sample by pipetting up and down.
(e) Dispense 12 μL of DNA/PCR buffer into each of the wells containing primer, being careful to lay the drop on the side of the well near the top. Do not allow the pipet tip to come in contact with the well contents.
(f) Aliquot 8 μL of the remaining Taq/PCR mix in the 1.5 mL tube into each of the control wells.
(g) Aliquot 5 μL of the positive control Macaca mulatta gDNA at 15 ng/μL into the appropriate wells.
(h) Firmly seal the tray or tubes.
(i) Vortex sealed tray or strip tubes and pulse spin down.
(j) Place on thermocycler and run program.
Thermocycling conditions:
96°C initial denaturation for 1 min
6 cycles
96°C denaturation for 25 s
67.9°C annealing for 50 s
72.0°C elongation for 45 s
6 cycles
96.0°C denaturation for 25 s
66.4°C annealing for 50 s
72.0°C elongation for 45 s
5 cycles:
96°C denaturation for 25 s
66.0°C annealing for 60 s (during which annealing temp is decreased 1.0°C for each of the five cycles)
72.0°C elongation for 45 s
16 cycles:
96.0°C denaturation for 25 s
63.0°C annealing for 50 s
72°C elongation at 45 s
72°C final extension for 10 min
25°C terminal hold
2. Analysis by gel electrophoresis
When cycling is completed, reactions are analyzed by gel electrophoresis using SB gels and buffer.
(a) Make a 2% agarose gel with 0.1 μg/mL of ethidium bromide in 0.5X SB.
| SB Buffer (BioTechniques 36:214-216, Feb 2004) | 100 bp DNA ladder (diluted) |
| 9 L milliQ water | 500 μL stock DNA ladder |
| 11.3 mL 10 M NaOH | 500 μL H2O |
| 1035 mL 0.5 M H3BO3 | Store at room temperature |
| Store at room temperature |
(b) Load 4 μL of DNA ladder into the first well of each lane of the gel.
(c) Load all 24 μL of the samples into wells.
(d) Run gel at 230 V for approximately 17 minutes.
(e) After running, take a picture with AlphaImager program (UV transillumination).
Protocol reference: Kaizu M, Borchardt, GJ, Glidden CE, Fisk DL, Loffredo JT, Watkins DI, Rehrauer WM. Molecular typing of major histocompatibility complex class I alleles in the Indian rhesus macaque which restrict SIV CD8+ T cell epitopes. Immunogenetics. 2007 Sep:59(9):693-703. Epub 2007 Jul 20.PMID: 17641866
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