Bitan:genotyping

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Genotyping

DNA Extraction

1. Weigh tail samples and confirm they are under 25μg, place in microcentrifuge tube 2. Add 100μL Buffer ATL from QIAamp DNA Mini Kit 3. Add 20μL proteinase K, mix by vortexing 4. Incubate in overnight at 56°C 5. Add 200μL Buffer AL, vortex on low for 15s 6. Incubate at 70°C for 10min 7. Add 200μL ethanol (96-100%) and mix by vortexing on low for 15s 8. Apply mixture to QIAamp minispin column and centrifuge at 6000XG for 1min 9. Place column in clean tube and add 500uL Buffer AW2 and centrifuge at 20,000XG for 3min 10. Place column in new tube and centrifuge at full speed for 1min 11. Place column in clean 1.5mL microcentrifuge tube 12. Add 200μL Buffer AE or distilled water 13. Incubate at room temperature for 5min 14. Centrifuge at 6,000XG for 1min 15. Repeat steps (12-14) twice for best results

PCR

1. Place a PureTaq Ready –To-Go PCR bead in a 1.5μL microcentrifuge tube 2. Add 2.5μL of forward primer and 2.5 μL backward primer 3. Add 11μL DNA 4. Add 9μL autoclaved water 5. Place sample in PCR machine and create a procedure for the according genes: initialization step for Taq needs hot start PS1: denature:95°C 5min, number of cycles: 42, 95°C for 30s denature, 61°C for 30s anneal, 72°C for 30s elongation, final 72°C for 5min final elongation APP/Tau: denature 95°C for 5min, number of cycles 35, 95°C for 30s, 52°C for 30s, 72°C for 50s, final 72°C for 8min 6. don’t allow PCR machine to hold samples at 10C overnight, it will explode 7. Run a tube with primers and bead only (no mouse dna) as control

BSTE II Digestion for PS1 samples

1. Add 10μL PCR DNA, 3μL 10XFast Digest Buffer, 3μL Fast Digest Eco911 enzyme, 14μL water a. For 30ul (22ul nuclease free water, 2ul 10x fast digest buffer, 5ul unpurified pcr product (~2ug), 1ul fast digest enzyme) 2. Run a non digested PS1 sample as control of digestion

1% Agrose Gel electrophoresis

1. make 1% gel, can’t make higher % because harder to melt later 2. 300ml Fresh 1x TBE + 3g Agrose → dissolve to a clear solution by microwaving in sealed glass jar, 45-60sec at a time, then mixing, opening and mixing, probably will take 2-4min, let cool in hood to ~50C (very hot to touch but not burning). If it cools too much, will see lines/waves in the liquid, reheat because gel won’t be homogenous. a. use 50ml tube to measure 300ml cuz grad cyl not clean enough b. be VERY careful opening jar and do it in the hood because the liquid may boil as soon as the pressure on it is released 3. add 5ul Ethidium bromide (stock at 1% w/v= 10u/ul, carcinogen) per 100ml of gel (thus need 15ul) (EtBr under hood by printer) 4. Pour gel in to cast, can put in fridge to solidify faster, then take off red ends and put in gel box wells towards black (see 7b below) a. Pouring gel needs 4 pieces: clear bottom, 2 red ends, comb b. Put brush in to gel such that knobs are towards center so it gives more room btwn comb and red side so when take out comb, gel doesn’t break 5. Add 2ul 6x orange loading dye to 10ul of sample (can load up to 15-30?ul/well) and pipette up and down to mix a. Store rest of the pcr product at -20C 6. Load all 12 ul of sample and 10ul of ladder in to gel 7. Run gel for 10min at 90V then 2hrs at 30V or 3hrs at 25V a. Maybe 40min at 70V (100V is no good) b. Black is neg, red is pos, DNA is neg so goes towards red/pos, put brush/wells by the black c. Put lid on, attach wires, turn on, put knob on min, DC start, turn volt knob to 100V for 10min, check for bubbles (bubbles on black side because 2 hydrogens being released) d. If leave gel run too long, the DNA moving away from the black side and the EtBr moving away from the red side will cross and samples will get destained and won’t see bands 8. visualize with UV Transilluminator a. slide gel on to middle of platform and get rid of bubbles underneath b. fluurchem FC2, acquire, EtBr filter position #2, transillum UV, reflective UV, speed/res normal. c. Zoom ROI, focus, put top knob at 1.8, lower knob used to focus on MW ladder band, acquire image d. File save, edit activation crop, make box, edit crop, contrast adjustment revers e. File, save modified f. Clean UV platform with EtOH after done. g. Gel should be saved and wasted specifically cuz of the EtBr

solutions

10x TBE (Tris Borate EDTA) 108 g Tris Base (final conc 89mM) 55 g Boric Acid (final conc 89mM) 40 ml 0.5M EDTA, ph 8.0 (final conc 2mM) Bring up to 1L with autoclaved water

TE buffer Final conc for 100ml: 10mM Tris ph8 (10ml of 100mM Tris ph8) 1mM EDTA (20ml of 5mM EDTA) 10mM NaCl (5ml of 200 mM NaCl) 65 ml water up to 100ml

STE For 500ml 50ml of 1M Tris (MW 121.14) ph 8.5 for final conc 100mM 5ml of EDTA (MW 372.2) 0.5M for final conc 5mM 10ml of 10% SDS (MW 288.38) for final conc 0.2% 20ml of 5M NaCl (MW 58.44) for final conc 200mM 415ml water

OR 50 ml of 100mM Tris ph 8.5 for final conc of 10mM 5ml of 5mM EDTA for final conc of .005mM 10ml of .2% SDS for final conc of .004% 20ml of 200mM NaCl for final conc of 8mM 415ml water

Primers are at 100uM when reconstituted (100pmol/1ul), to make 20uM take 20ul primer and add 80ul TE buffer = 100ul. To reconstitute primers use 10x the nmoles in ul (50nM needs 500ul of TE)

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