Lidstrom:Genomic DNA extraction: Difference between revisions
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Back to [[Lidstrom:Protocols|Protocols]] | Back to [[Lidstrom:Protocols|Protocols]] | ||
Genomic DNA | == Genomic DNA minikit extraction PROTOCOL == | ||
* From Marina K. | |||
Please wear gloves at all times | * Please wear gloves at all times | ||
# Add 180 µl Buffer ALT to a clean 1.5ml tube | # Add 180 µl Buffer ALT to a clean 1.5ml tube | ||
# Add 20 µl proteinase K and mix | # Add 20 µl proteinase K and mix | ||
# Scoop cell biomass from Petri-plate and add it to ATL/ proteinase K mixture. Mix well; | # Scoop cell biomass from Petri-plate and add it to ATL/ proteinase K mixture. Mix well; | ||
Line 22: | Line 22: | ||
# Your DNA sample is now in the 1.5ml tube (should be 200ul). Discard the Spin Column. | # Your DNA sample is now in the 1.5ml tube (should be 200ul). Discard the Spin Column. | ||
# Measure DNA concentration on nanoDrop | # 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 | |||
# In a 15 ml tube resuspend cell pellet in 5 mL lysis buffer | |||
# Add 100 ul of 100 mg/ml proteinase K and 100 ul of 50 mg/ml RNase A | |||
# Mix gently and incubate 6 hrs - overnight in a 50°C water bath | |||
# Add 5 ml phenol/chlorofom/isoamyl alcohol (IAA) (25:24:1) pH 8 solution and mix by gentle inversion for 10 min at room temp | |||
# Centrifuge at 4000 RPM, 4°C, for 15 min | |||
# 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 | |||
# Add an equal amount of chloroform/IAA (24:1) solution and mix by gentle inversion for 10 min at room temp | |||
# Centrifuge at 4000 RPM, 4°C, for 15 min | |||
# Using a wide-bore tip, transfer the upper aqueous layer (should be ~ 5 ml) to a new 15 ml tube | |||
# Repeat steps 7-9 two more times | |||
# To precipitate gDNA from the aqueous layer, add 1/10th the volume of 3M sodium acetate pH 5.5 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 | |||
# Using a wide-bore tip, collect gDNA and transfer to a new 15 ml tube | |||
# add 5 ml 70% ethanol to wash gDNA, and centrifuge | |||
# Discard supernatant and centrifuge again (want to remove as much ethanol as possible) | |||
# Pipette off any residual liquid and dry sample for 15 min at RT with cap off | |||
# Resuspend gDNA in 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 | |||
# Check quality of gDNA by NanoDrop and by electrophoresis |
Revision as of 12:23, 26 February 2013
Back to Protocols
Genomic DNA minikit extraction PROTOCOL
- From Marina K.
- Please wear gloves at all times
- Add 180 µl Buffer ALT to a clean 1.5ml tube
- Add 20 µl proteinase K and mix
- Scoop cell biomass from Petri-plate and add it to ATL/ proteinase K mixture. Mix well;
- Incubate at 56oC for 3 h or overnight
- Add 200 µl Buffer AL to the sample, mix by pulse-vortexing for 15 s, and incubate at 70 °C for 10 min.
- Briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from inside the lid.
- Add 200 µl ethanol to the sample, and mix by pulse-vortexing for 15 s.
- 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.
- 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.
- Place the SpinColumn in a clean 2 ml collection tube and discard the collection tube containing the filtrate.
- 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.
- Place the SpinColumn in a clean 2 ml collection tube and discard the collection tube containing the filtrate
- Centrifuge at full speed (14,000 rpm) for 2 min.
- Place the SpinColumn in a clean 1.5 ml tube and discard the collection tube
- 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.
- Your DNA sample is now in the 1.5ml tube (should be 200ul). Discard the Spin Column.
- 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
- In a 15 ml tube resuspend cell pellet in 5 mL lysis buffer
- Add 100 ul of 100 mg/ml proteinase K and 100 ul of 50 mg/ml RNase A
- Mix gently and incubate 6 hrs - overnight in a 50°C water bath
- Add 5 ml phenol/chlorofom/isoamyl alcohol (IAA) (25:24:1) pH 8 solution and mix by gentle inversion for 10 min at room temp
- Centrifuge at 4000 RPM, 4°C, for 15 min
- 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
- Add an equal amount of chloroform/IAA (24:1) solution and mix by gentle inversion for 10 min at room temp
- Centrifuge at 4000 RPM, 4°C, for 15 min
- Using a wide-bore tip, transfer the upper aqueous layer (should be ~ 5 ml) to a new 15 ml tube
- Repeat steps 7-9 two more times
- To precipitate gDNA from the aqueous layer, add 1/10th the volume of 3M sodium acetate pH 5.5 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
- Using a wide-bore tip, collect gDNA and transfer to a new 15 ml tube
- add 5 ml 70% ethanol to wash gDNA, and centrifuge
- Discard supernatant and centrifuge again (want to remove as much ethanol as possible)
- Pipette off any residual liquid and dry sample for 15 min at RT with cap off
- Resuspend gDNA in 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
- Check quality of gDNA by NanoDrop and by electrophoresis