EXPERIMENT 2
Mosquito DNA Extraction Protocol
MATERIALS
Mosquito DNA Extraction Protocol
Lab notebook
Lab coat
Gloves
56C water bath with tube racks
Frozen mosquitoes
95% ethanol
Eppendorf tubes
Tube racks
Permanent marker
Micropipette, P-1000 with tips
Micropipette, P-200 with tips
Micropipette, P-20 with tips
Beaker for used tips
ATL buffer
Sterile forceps
MilliQ water
Proteinase K
Microcentrifuge
PROCEDURES
Part A. Get Ready
1. This protocal has been adapted from QIAGEN’s “Purification of total DNA from insects using the
DNeasy Blood & Tissue Kit (Qiagen.com, Cat. No. 69504).
2. Put on a lab coat and gloves and wear them throughout all procedures.
3. As specified in the Laboratory Notebook Preparation handout, setup your lab notebook for
Experiment 2.
4. Except where noted, all procedures are carried out at room termperature.
5. Prior to your lab, your instructor will have a water bath warmed to 56C.
6. Prior to lab, your instructor will have added ethanol to the AW1 and AW2 buffers as specified on
the reagent bottles.
7. The ATL and AL buffers may form solid precipitates upon storage. Observe those buffers. If a
precipitate is present, place the tube in a tube rack in the 56C water bath and allow the tube to
warm until the precipitates have dissolved fully.
Part B. Grind and Lyse a Mosquito
1. Label an eppendorf tube as 95% ethanol and pipette 500
2.
3. Bringing a sterile forceps and tube rack containing the tube of 95% ethanol, go to the -20
Freezer A in Room 109.
4. In the freezer, find the eppendorf tube labeled with your group’s number.
5. Quickly, use the forceps to remove one mosquito and place it into the 95% ethanol tube. DO
NOT FREEZE THAW mosquitoes repeatedly!!! Work quickly when removing the insect from the
tube to preserve the rest in the tube for future work.
6. Return to your lab bench with your mosquito.
7. Use a sterile forceps to remove the wings from the mosquito. The wings may be discarded.
8. Use a sterile forceps to remove the wingless mosquito from the 95% ethanol tube and rinse the
95% ethanol thoroughly off of the mosquito with sterile milliQ water. You may do the rinsing
into the rinse beaker.
9. Add the rinsed wingless mosquito to tube ATL.
10. In the ATL tube, grind the insect thoroughly using a pestle.
11.
L.
12. Mix the contents of tube ATL thoroughly by pulse-vortexing for 5 – 10s.
13. Place tube ATL into a tube rack in the 56C water bath.
14. Incubate tube ATL for 1 – 3 hours or overnight. If an overnight incubation is chosen, the
remainder of this procedure MUST be completed the next day.
15. If the incubation is not overnight, set a timer for the desired incubation length.
Part C. Extract the DNA
1. During the incubation:
a. Label an eppendorf tube as AE1.
b. Pipette 220
c. Place tube AE1 in a tube rack in the 56C water bath.
d. Label an eppendorf tube as premix.
e.
f.
-100%) into the premix tube.
Mix the contents of the premix tube thoroughly by pulse-vortexing for 5 – 10s.
g. Label three collection tubes as spin 1, spin 2, and spin 3.
h. Place a DNeasy Mini spin column into spin tube 1.
2. After the incubation, remix the contents of the premix tube thoroughly by pulse-vortexing for 5
– 10s.
3. Remove tube ATL from the 56C water bath.
4. Pipette the 400
by pulse vortexing for 5 – 10s.
a. A white precipitate may form or the mixture may appear gelatinous. If so, vortex the
tube vigorously before the next step.
5. Pipette the mixture, including any precipitate, from step 4 above into the DNeasy Mini spin
column in spin tube 1.
6. Centrifuge spin tube 1 at 8000rpm for 1 minute.
7. Remove the DNeasy Mini spin column from spin tube 1 and place it into spin tube 2.
8.
the DNeasy Mini spin column in spin tube 2.
9. Centrifruge spin tube 2 at 8000rpm for 1 minute.
10. Remove the DNeasy Mini spin column from spin tube 2 and place it into spin tube 3.
11.
12. Centrifruge spin tube 3 at 14000rpm for 3 minutes.
13. During that spin, label an eppendorf tube as AE2.
14. Carefully remove the DNeasy Mini spin column from spin tube 3 so that it does NOT come into
contact with the flow-through and place it into the AE2 tube.
a. If the DNeasy Mini spin column does come into contact with the flow through, repeat
the last spin.
15. Pipette 100
AE2.
16. Incubate tube AE2 at room temperature for 1 minute.
17. Centrifuge tube AE2 at 8000rpm for 1 minute.
18. Remove the DNeasy Mini spin column from tube AE2 and place it into tube AE3.
19. Place tube AE2 on ice.
20.
AE3.
21. Incubate tube AE3 at room temperature for 1 minute.
22. Centrifuge tube AE3 at 8000rpm for 1 minute.
23. Remove the DNeasy Mini spin column from tube AE3.
24. Place tube AE3 on ice.
25. Discard all tubes except tubes AE2 and AE3.
6.) Place DNeasy Mini spin column in a clean 1.5ml microcentrifuge tube. Add 100ul of pre-warmed
AE Buffer directly onto the DNeasy membrane. Incubate at room temperature for 1 minute,
then centrifuge for 1 minute. Place the column in a fresh microcentrifuge tube and repeat this
process once for a total of 200ul in two separate tubes. Put tubes on ice or in the fridge while
doing the rest of the steps.