McClean: Genomic DNA Prep (Bust 'n' Grab Protocol)

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(New page: <!-- COPY EVERYHING BELOW HERE TO START YOUR OWN PROTOCOL! --> ==Overview== This protocol is for rapidly isolating genomic DNA from an overnight culture of yeast. This protocol was adap...)
(Notes)
Line 32: Line 32:
<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->
<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!  Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!  Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
-
human carcinogen and reproductive toxin
 
-
Affects the central nervous system (depressant)
 
-
*[[User:Megan N McClean|Megan N McClean]] 18:25, 29 June 2012 (EDT) Chloroform is a suspected human carcinogen and reproductive toxin.  If affects the central nervous system acting as well as a depressant.  Exercise appropriate caution when handling chloroform.  Wear gloves, a lab coat, splash goggles and work in the fume hood.  Gloves should be Polyvinyl alcohol (PVA) or laminate barrier (Silver Shield®), not nitrile or latex.  Use caution when centrifuging chloroform.  If you suspect that a tube has opened or broken while centrifuging wait 10 minutes for the aerosolized chloroform to settle out before open the centrifuge in the fume hood.
 
-
*[[User:Megan N McClean|Megan N McClean]] 17:48, 29 June 2012 (EDT) I use this protocol because it is easy and quick.  For PCR I almost never measure the concentration of genomic DNA when I am done, I just dilute 1:100 in water and use 1μL for 50μL for PCR.  The concentration of genomic DNA in the PCR reaction of course does matter, and if your preps are off you will need to either go back and measure how much genomic DNA you actually have or (what I usually do) try different dilutions (1:10 or 1:1000 usually works for me if 1:10 fails).
 
 +
*[[User:Megan N McClean|Megan N McClean]] 18:25, 29 June 2012 (EDT) Chloroform is a suspected human carcinogen and reproductive toxin.  It is a depressant and affects the central nervous system.  Exercise appropriate caution when handling chloroform.  Wear gloves, a lab coat, splash goggles and work in the fume hood.  Gloves should be Polyvinyl alcohol (PVA) or laminate barrier (Silver Shield®), not nitrile or latex.  Use caution when centrifuging chloroform.  If you suspect that a tube has opened or broken while centrifuging wait 10 minutes for the aerosolized chloroform to settle out before open the centrifuge in the fume hood.
 +
*[[User:Megan N McClean|Megan N McClean]] 17:48, 29 June 2012 (EDT) I use this protocol because it is easy and quick.  For PCR I almost never measure the concentration of genomic DNA when I am done, I just dilute 1:100 in water and use 1μL for 50μL for PCR.  The concentration of genomic DNA in the PCR reaction of course does matter, and if your preps are off you will need to either go back and measure how much genomic DNA you actually have or (what I usually do) try different dilutions (1:10 or 1:1000 usually works for me if 1:10 fails).
==References==
==References==

Revision as of 18:25, 29 June 2012


Contents

Overview

This protocol is for rapidly isolating genomic DNA from an overnight culture of yeast. This protocol was adapted from Harju, et al (2004). Please see the reference below and cite it if you use this protocol.

Materials

  • Overnight yeast culture (grown for 20-24h) in YPD
  • Dry ice-ethanol bath
  • Lysis buffer (2% Triton X-100, 1% SDS, 100mM NaCl, 10mM Tris-HCL, pH 8.0, 1mM EDTA, pH 8.0)
  • Chloroform
  • 100% Ethanol (ice-cold, put in -20°C before you start this protocol)
  • 70% Ethanol
  • TE (10mM Tris, pH 8.0, 1mM EDTA, pH 8.0)
  • (optional) RNase cocktail (Ambion)

Protocol

  1. Transfer 1.5 ml of liquid culture of yeast grown for 20 – 24 h at 30°C in YPD into a microcentrifuge tube. Pellet cells by centrifugation at 20,000 × g for 5 minutes.
  2. Add 200 μl of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0).
  3. Immerse tubes in a dry ice-ethanol bath for 2 minutes, transfer to in a 95°C water bath for 1 minute. Repeat; vortex 30 seconds.
  4. Add 200 μl of chloroform; vortex 2 minutes.
  5. Centrifuge 3 minutes, room temperature, 20,000 × g.
  6. Transfer the upper aqueous phase to a microcentrifuge tube containing 400 μl ice-cold 100% ethanol. Mix by inversion or gentle vortexing.
  7. Incubate at room temperature, 5 minutes. Alternatively, precipitate at -20°C to increase yield.
  8. Centrifuge 5 minutes, room temperature, 20,000 × g. Remove supernatant with a pulled Pasteur pipette by vacuum aspiration.
  9. Wash the pellet with 0.5 ml 70% ethanol, spin down as described in step 8 above. Remove supernatant.
  10. Air-dry the pellets at room temperature or for 5 minutes at 60°C in a vacuum dryer.
  11. Resuspend in 25–50 μl TE [10 mM Tris (pH 8.0), 1 mM EDTA (pH 8.0)] or water. Samples obtained directly from plates should be resuspended in a 10 μl volume, because the yield will be smaller. 0.25 μl RNase cocktail (Ambion) should be added to the samples used for Southern blot hybridization (final concentration 0.125 U RNAse A, 5 U RNase T1).


Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.


  • Megan N McClean 18:25, 29 June 2012 (EDT) Chloroform is a suspected human carcinogen and reproductive toxin. It is a depressant and affects the central nervous system. Exercise appropriate caution when handling chloroform. Wear gloves, a lab coat, splash goggles and work in the fume hood. Gloves should be Polyvinyl alcohol (PVA) or laminate barrier (Silver Shield®), not nitrile or latex. Use caution when centrifuging chloroform. If you suspect that a tube has opened or broken while centrifuging wait 10 minutes for the aerosolized chloroform to settle out before open the centrifuge in the fume hood.
  • Megan N McClean 17:48, 29 June 2012 (EDT) I use this protocol because it is easy and quick. For PCR I almost never measure the concentration of genomic DNA when I am done, I just dilute 1:100 in water and use 1μL for 50μL for PCR. The concentration of genomic DNA in the PCR reaction of course does matter, and if your preps are off you will need to either go back and measure how much genomic DNA you actually have or (what I usually do) try different dilutions (1:10 or 1:1000 usually works for me if 1:10 fails).

References

  • Harju, S and H. Fedosyuk and KR Peterson (2004) Rapid isolation of yeast genomic DNA: Bust n' Grab BMC Biotechnol 4:8

Contact

or instead, discuss this protocol.

Personal tools