Oct 06, 2015
DNA Extraction for college laboratory setting
- James M. Burnette III,
- Susan R. Wessler
- Genetics
External link: http://www.genetics.org/content/193/2/367.full
Protocol Citation: James M. Burnette III, Susan R. Wessler 2015. DNA Extraction for college laboratory setting. protocols.io https://dx.doi.org/10.17504/protocols.io.dwu7ev
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: September 26, 2015
Last Modified: March 22, 2018
Protocol Integer ID: 1716
Abstract
This protocol is based on one described by Li et al. (2010) and has been modified to work in a college laboratory setting. The protocol is from:
James M. Burnette III andSusan R. Wessler (2013) Transposing from the Laboratory to the Classroom to Generate Authentic Research Experiences for UndergraduatesGenetics193:367-375; doi:10.1534/genetics.112.147355
Students need approximately 1.5 hours to extract DNA from up to five samples and the protocol can be carried out over several class periods by stopping at steps 6 and 9. Please see the full manuscript for additional details.
Guidelines
This protocol is based on one described by LI et al. (2010) and has been modified to work in a college laboratory setting. Students need approximately 1.5 hours to extract DNA from up to five samples and the protocol can be carried out over several class periods by stopping at steps 6 and 9.
Materials list:
Extraction Buffer (100 mM Tris, pH 8.0, 50 mM EDTA and 500 mM NaCl)
10% SDS (sodium dodecyl sulfate)
5M KOAc (Potassium Acetate)
1 5 cm by 5 cm piece of Miracloth (Calbiochem, La Jolla, CA)
100% Isopropanol
70% Ethanol
Sterile water
Ice
Liquid nitrogen
65˚C heating block
Sterile 1.5 ml tubes (2 for each prep)
Mortar and pestle
All chemicals were purchased from Fisher Scientific.
Label one tube for each plant.
Harvest 2-3 seedlings and place in a mortar. Fill with about 50 ml of liquid nitrogen. Grind tissue with pestle.
Add 1 ml of extraction buffer to the tube.
Add 120 µl of 10% SDS. Mix by inverting.
Note
If preparing more than one sample, prepare each sample to this step and place on ice.
Incubate tube(s) at 65 ˚C for 20 minutes.
00:20:00
Add 300 µl 5M KOAc. Mix well by inverting several times (important!), then place on ice 5 minutes.
00:05:00
Note
Stopping point: Samples can be frozen for a future class period. Thaw samples before starting with step 7.
Centrifuge for 5 minutes at >12,000 rpm. Label a second tube.
00:05:00
Pass 700 µl of the supernatant through a miracloth funnel into the second tube.
Add 600 µl of isopropanol. Mix the contents thoroughly by inverting.
Note
Stopping point: Samples can be frozen for a future class period.
Spin for 5 minutes at 14,000 rpm.
00:05:00
Carefully pour off and discard the supernatant. Use a P20 set to 20 µl to remove the remaining drops of liquid without disturbing the DNA pellet.
Add 500 µl of 70% ethanol and flick the tube until the pellet comes off the bottom.
Spin 5 minutes.
00:05:00
Pour off the ethanol. Use a P20 set to 20 µl to remove the remaining drops without disturbing the pellet.
Leave the tube open on the bench to air dry for 5-10 minutes.
00:05:00
Resuspend the DNA in 50 µl TE and incubate at room temperature for 5 minutes for complete resuspension. Samples should be frozen for storage.
00:05:00