Lidstrom:Genomic DNA extraction

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Genomic DNA minikit extraction PROTOCOL

• From Marina K.
• Please wear gloves at all times

1. Add 180 µl Buffer ALT to a clean 1.5ml tube
2. Add 20 µl proteinase K and mix
3. Scoop cell biomass from Petri-plate and add it to ATL/ proteinase K mixture. Mix well;
4. Incubate at 56oC for 3 h or overnight
5. Add 200 µl Buffer AL to the sample, mix by pulse-vortexing for 15 s, and incubate at 70 °C for 10 min.
6. Briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from inside the lid.
7. Add 200 µl ethanol to the sample, and mix by pulse-vortexing for 15 s.
8. Carefully apply the mixture from step 4 (including the precipitate) to the Spin Column (in a 2 ml collection tube). Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the Spin Column in a clean 2 ml collection tube (provided),and discard the tube containing the filtrate.
9. Carefully open the Spin Column and add 500 µl Buffer AW1. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min.
10. Place the SpinColumn in a clean 2 ml collection tube and discard the collection tube containing the filtrate.
11. Carefully open the Spin Column and add 500 µl Buffer AW2 without wetting the rim. Close the cap and centrifuge at full speed (14,000 rpm) for 3 min.
12. Place the SpinColumn in a clean 2 ml collection tube and discard the collection tube containing the filtrate
13. Centrifuge at full speed (14,000 rpm) for 2 min.
14. Place the SpinColumn in a clean 1.5 ml tube and discard the collection tube
15. Carefully open the Spin Column and add 100 µl Buffer AE. Incubate at room temperature for 2 min, and then centrifuge at 12 000 rpm for 1 min. Repeat this step.
16. Your DNA sample is now in the 1.5ml tube (should be 200ul). Discard the Spin Column.
17. Measure DNA concentration on nanoDrop

Comments

• Use approximately "a booger" worth of cells
  • If your cells are on a plate, wash them off with their growth medium and pellet in a centrifgue.
• Aaron Puri incubated cells + ALT + proteinase K for 6 hours and got 40 ng/uL.

Genomic DNA phenol/chloroform extraction PROTOCOL

• From Marina K.
• Please wear (nitrile) gloves at all times!
• All phenol/chloroform steps should be conducted in a fume hood
• Special equipment or reagents needed:

Mini LabRoller for gentle inversion

DNA lysis buffer

10 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 2% (w/v) SDS

1000 ul pipette tips with the points cut off to make a "wide-bore" tip (prevents shearing of chromosomal DNA)

• Cells can be collected from plates or liquid culture

If plates, use 1-2 ml liquid medium and a disposable loop or small serilogical pipette to lift up and suspend cells

If liquid culture, centrifuge in 50 ml tubes before transferring pellet to 15 ml tube

1. In a 15 ml tube resuspend cell pellet in 5 mL lysis buffer (FC bench)
2. Add 100 ul of 100 mg/ml proteinase K (FC bench) and 100 ul of 50 mg/ml RNase A (FC bench)
3. Mix gently and incubate 6 hrs - overnight in a 50°C water bath (Frances puts in the 55 deg C incubator)
4. Add 5 ml(equal volume) phenol/chlorofom/isoamyl alcohol (IAA) (25:24:1) pH 8 solution (fridge in the RNA room) and mix by gentle inversion for 10 min at room temp
5. Centrifuge at 4000 RPM, 4°C, for 15 min
6. Using a wide-bore tip, transfer the upper aqueous layer (should be ~ 5 ml) to a new 15 ml tube
   • Avoid touching the interphase! This is where the proteins and cell debris collect; usually appears as a thick layer of white, but can be more yellow and thinner depending on the species
7. Add an equal amount of chloroform/IAA (24:1) solution and mix by gentle inversion for 10 min at room temp
8. Centrifuge at 4000 RPM, 4°C, for 15 min
9. Using a wide-bore tip, transfer the upper aqueous layer (should be ~ 5 ml) to a new 15 ml tube
10. Repeat steps 7-9 two more times (Frances skips this, says she tried and it didn't make a difference)
11. To precipitate gDNA from the aqueous layer, add 1/10th the volume of 3M sodium acetate pH 5.5(Frances' protocol uses 0.3M) and 2.5 volumes of ethanol
    • Holding the tube between your pointer fingers, gently rock the tube up and down; gDNA will spool out of solution and appear as fluffy white-ish gob
12. Using a wide-bore tip, collect gDNA and transfer to a new 15 ml tube
    • Frances just centrifuges for 30 min at 5000 rpm, 4°C, then discards the supernatant
13. add 5 ml 70% ethanol to wash gDNA, and centrifuge 5 min @ 5000 rpm, 4 °C
14. Discard supernatant and centrifuge again (want to remove as much ethanol as possible)
15. Pipette off any residual liquid and dry sample for 15 min at RT with cap off
16. Resuspend gDNA in (0.25-)0.5 ml TE buffer
    • Full resuspension of gDNA can take a couple days in the fridge; NanoDrop measurement of concentration will not be anywhere near accurate until gDNA has been completely resuspended
17. Check quality of gDNA by NanoDrop and by electrophoresis
    • 260/280 ~1.8-2.0