Protocol
DNA Extraction
HOMOGENIZATION/LYSIS
1. Weigh out 100 mg needle tissue or terminal bud, terminal bud preferred
2. Using sterile technique, slice needles into ~1cm pieces
3. Flash freeze and emulsify in mortar and pestle using liquid nitrogen [Ask Mimi if LN2 is empty, takes 1 day]
4. Transfer ground tissue to 1.7 ml eppi tube
5. Add 1,000 ul of lysis buffer (2x CTAB, 2% PVP, recipe found in CCDB Protocols)
6. Add 10 ul ProteinaseK
7. Incubate overnight at 65 degrees Celsius, 400 RPM in thermo mixer
DNA EXTRACTION
1. Use MO BIO Kit, follow instructions
QC
1. Use nanodrop to measure concentration and quality
2. Make sure to blank w/ elution buffer
3. Record 260/230, 260/280, and mg/ml
4. Run 2ul in gel, to see fragment distribution (nonsheared high MW, ideal)
PCR of Microsatellites
MASTER MIX, Qiagen (small box in freezer)
Qiagen Mastermix, n=1 5 ul
F primer, labelled (10 uM) 0.5 ul
R primer (5 uM) 1 ul
Q sol 2 ul
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8.5 ul
1. Prepare Master Mix for n+1, n=number of samples
2. Add 8.5 ul to pcr strip tube
3. Add 2ul of DNA, mix. Total volume = 10.5 ul
4. Run thermal cycler, Program > Daniel > Potter
5. Store in cold room
Thermal Cycler Conditions (Potter PCR)
94*C 15 min
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94*C 30 s ]
60*C 30 s ] 4x
72*C 1 min ]
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94*C 30 s ]
57*C 30 s ] 4x
72*C 1 min ]
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94*C 30 s ]
55*C 30 s ] 30x
72*C 1 min ]
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Extend 72*C, 7 min
Submitting Samples to CORE for sequencing
1. Let me know the PI name and account number that will need to be charged for the runs. The cost per sample (capillary run) is $2 for on campus accounts
2. Pick up 96 well submission plates from my lab (no charge) *Located above blue incubator, plastic bag, clear semi-skirted w/ barcode
3. Fill out the attached Excel template file with sample names when you have a plate to run. Plate should be filled by column in the order of the Excel template. You can submit anywhere from 1 to 96 samples. Minimum charge of 16 samples will apply.
4. Aliquot your diluted (1:10) PCR product into the appropriate wells of the plate and seal with plastic or aluminum film. I’ll add Hi-Di formamide and size standard when I receive your plate. **Note if bands are faint, don't dilute, submit 2 ul
5. Email the sample list back to me and bring your plate over – I can typically run the plate the same night with data available the next morning. Also let me know the expected sizes of your PCR fragments, and the fluorescent tag they are labeled with.
Excel sheet for fragment analysis
