BIOLOGY 40-Protocol #1
Summer, 2007
Name______________________________
Genotype____________
Protocol 1: DNA isolation from Drosophila melanogaster
Fly DNA mini prep.
1. Write the genotype of the flies you will be working with in your notebook and on this
protocol sheet.
2. Transfer your live flies from the vial with food into a clean empty vial.*
3. Place the fresh vial of flies into the freezer (at -20oC) or into a container of wet ice to
put the flies asleep.
4. Transfer 10-20 of the sleeping flies to a blue microcentrifuge tube (be sure the tube
will fit the pellet pestle for grinding. Place the filled tube on crushed dry ice. Be sure
the ice covers the tube completely and that there are no air spaces around the tube. *
5. Put gloves on at this point and use throughout the protocol when working with
the flies and the solutions.
6. Remove the frozen tube of flies from the dry ice and add 100 l of Grinding Buffer
to the tubes. Immediately use the pellet pestle to grind the flies into the solution.
Note: The Grinding Buffer contains 5% sucrose, 80 mM NaCl, 80mM Tris pH8.5, 0.5%
SDS, and 50mM EDTA. The sucrose prevents oxidation of the compounds in the fly
cells and allows the cells to help take up the solutions. The SDS is a detergent that opens
the phospholipid bilayer of the plasma membrane of the cells. The EDTA chelates the
Mg++ and Ca++ ions from the tissues. These ions function as cofactors that allow
degradative enzymes called nucleases to cut up the nucleotides. By removing the ions
the nucleases cannot cut up the DNA and RNA.
7. Grind the flies quickly and thoroughly by grinding the pellet pestle against the sides
and bottom of the microcentrifuge tubes until you have a homogeneous solution.
8. Remove the pellet pestle and rinse the ground matter from the flies by adding another
100 l of Grinding Buffer, pipetting the solution over the pellet pestle into the tube of
ground flies.
9. Refreeze the tube with the ground flies on dry ice for 5 minutes until the flies are
completely frozen.**
Modifications to the protocol (6/11/07)
* This step was omitted.
** Used -20oC freezer instead of dry ice.
BIOLOGY 40-Protocol #1
Summer, 2007
10. Remove from the dry ice and incubate in a water bath at 70°C for 30 minutes. Note:
You may shorten this interval by 15 minutes but the yield of DNA will be less.
11. Add 35 l of ice cold 8 M potassium acetate to the warm tube of ground flies and mix
thoroughly by flicking the tube gently with your finger. The potassium acetate
solution will cause the proteins to pellet out of solution.
12. Incubate the tube in an ice bath for 30 minutes. (Again this step may be shortened by
15 minutes but the quality of DNA will be reduced.)
13. Load the tubes in to the rotor of the microcentrifuge placing the hinge of the tube lid
towards the outer side of the rotor. Be sure to balance the tubes.
14. Centrifuge the tube in the microcentrifuge for 10 minutes at 14,000 RPM to pellet the
protein precipitate at the bottom of the tube.
15. Carefully remove the tube from the centrifuge and immediately remove the
supernatant by using a clean 100-200 l pipettor and gently pulling off the liquid. Be
sure you have not picked up any of the white precipitate in the solution.
16. Put the supernatant into a clean microcentrifuge tube and add 150l of isopropanol to
the tube. Mix by gently inverting the tube several times. Place in a rack for 5
minutes to allow the DNA to precipitate or store at -20oC overnight.
17. Spin the microcentrifuge tube for 10 minutes in order to pellet the precipitated DNA
at the bottom of the tube.
18. Remove the tube from the microcentrifuge tube and gently pour out the supernate
onto a clean paper towel. Be careful not to pour out the pellet.
19. Add 100 l of 80% ethanol to rinse off the DNA pellet and then spin in the
microcentrifuge at 14,000 RPM for 1 minute to once again pellet the DNA at the
bottom of the tube.
20. Repeat the ethanol wash step two times. Pour off the supernatant and allow the pellet
to air dry.
21. When the pellet is dry add 100 l of TE. Store the DNA solution in the
-20oC freezer.
Modifications to the protocol (6/11/07)
* This step was omitted.
** Used -20oC freezer instead of dry ice.
BIOLOGY 40-Protocol #1
Summer, 2007
SOLUTIONS PREPARED BY STUDENTS
50X TAE: Tris Acetate EDTA
 48.4 grams Tris base
 11.4 grams glacial acetic acid (pour into 50 ml conical tube in fume hood)
 7.44 grams EDTA
To reduce chances of contamination do not use tools (spatulas, spoons, etc.) to remove
solid reagents from their containers unless they have been sterilized. If sterile utensils are
not available, pour each solid reagent into the lid of its container and then pour slowly
into weigh boat until desired mass is obtained. If stir bars are used, they need to be
autoclaved. Most reagents will go into solution without using a stir bar.
Dilute to 100 ml with ddH20.
When reagents are in solution, dilute to 200 ml final volume
20X SSC (For Northern Blots)
To make 1 liter of solution you will need:
 173 g NaCl
 88.2 g Sodium citrate
 Add 850 ml ddH20
Adjust pH to 7.0 with 6 N HCl.
Do not place pH meter electrode in the stock solution, instead remove a small amount at a
time to check pH.
Dilute to 1000 ml with ddH20
Autoclave