Lidstrom:Genomic DNA extraction - Revision history

Nicole Smalley at 19:23, 26 February 2013

Nicole Smalley — Tue, 26 Feb 2013 19:23:34 GMT

←Older revision		Revision as of 19:23, 26 February 2013
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	# Measure DNA concentration on nanoDrop		# Measure DNA concentration on nanoDrop

-	== Comments==	+	=== Comments ===
	* Use approximately "a booger" worth of cells		* 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.		** 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.		* 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

Nicole Smalley: /* Genomic DNA extraction PROTOCOL */

Nicole Smalley — Tue, 26 Feb 2013 18:39:52 GMT

Genomic DNA extraction PROTOCOL

←Older revision		Revision as of 18:39, 26 February 2013
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	Back to [[Lidstrom:Protocols\|Protocols]]		Back to [[Lidstrom:Protocols\|Protocols]]

-	== Genomic DNA extraction PROTOCOL ==	+	== Genomic DNA minikit extraction PROTOCOL ==
	* From Marina K.		* From Marina K.
	* Please wear gloves at all times		* Please wear gloves at all times

Janet B. Matsen at 19:32, 20 February 2013

Janet B. Matsen — Wed, 20 Feb 2013 19:32:00 GMT

←Older revision		Revision as of 19:32, 20 February 2013
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	Back to [[Lidstrom:Protocols\|Protocols]]		Back to [[Lidstrom:Protocols\|Protocols]]

-	Genomic DNA ~~extractionPROTOCOL~~	+	== Genomic DNA extraction PROTOCOL ==
-	~~DNA extraction~~	+	* 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
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	# 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.

Janet B. Matsen at 17:53, 20 February 2013

Janet B. Matsen — Wed, 20 Feb 2013 17:53:53 GMT

←Older revision		Revision as of 17:53, 20 February 2013
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	Please wear gloves at all times		Please wear gloves at all times

-	# Add 180 µl Buffer ALT to a clean 1.5ml tube;l	+	# 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;

Janet B. Matsen at 17:50, 20 February 2013

Janet B. Matsen — Wed, 20 Feb 2013 17:50:18 GMT

←Older revision		Revision as of 17:50, 20 February 2013
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-	Genomic DNA extractionPROTOCOL 1	+	Back to [[Lidstrom:Protocols\|Protocols]]
		+
		+	Genomic DNA extractionPROTOCOL
	DNA extraction		DNA extraction
	Please wear gloves at all times		Please wear gloves at all times

Janet B. Matsen: New page: Genomic DNA extractionPROTOCOL 1 DNA extraction Please wear gloves at all times # Add 180 µl Buffer ALT to a clean 1.5ml tube;l # Add 20 µl proteinase K and mix # Scoop cell biomass fr...

Janet B. Matsen — Mon, 04 Feb 2013 23:47:03 GMT

New page: Genomic DNA extractionPROTOCOL 1 DNA extraction Please wear gloves at all times # Add 180 µl Buffer ALT to a clean 1.5ml tube;l # Add 20 µl proteinase K and mix # Scoop cell biomass fr...

New page

Genomic DNA extractionPROTOCOL 1
DNA extraction
Please wear gloves at all times

# Add 180 µl Buffer ALT to a clean 1.5ml tube;l
# 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