EXPERIMENT 2 – Mosquito DNA Extraction Protocol
MATERIALS
Mosquito DNA Extraction Protocol
Lab notebook
Glue stick
Lab coat
Gloves
Mosquito frozen at -20

C
Heat block prewarmed to 56

C
Eppendorf tubes, 26
Tube rack
Permanent marker
Micropipette, P-20 with tips
Micropipette, P-200 with tips
Micropipette, P-1000 with tips
Beaker for used pipette tips
Sterile forceps, 2 pairs
Rinse beaker, 50ml
Pestle
Vortex
Timer
DNeasy mini spin column
Collection tubes, 3
Microcentrifuge
Ice bucket filled with ice
CHEMICALS
95% ethanol, 500
 l
ATL buffer, 180
 l
Sterile milliQ water in squeeze bottle
Proteinase K, 20
 l
AE buffer, 250
 l
AL buffer, 200
 l
100% ethanol, 200
 l
AW1 buffer, 500
 l
AW2 buffer, 500
 l
1

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.
You will setup your lab notebook during an incubation break so for now, just follow this protocol.
4.
Locate all materials and chemicals listed above. Most will be at your lab bench.
5.
Except where noted, all procedures are carried out at room temperature.
6.
Prior to lab, your instructor will have a heat block warmed to 56

C.
7.
Prior to lab, your instructor will have added ethanol to the AW1 and AW2 buffers as specified on the reagent bottles.
8.
The ATL and AL buffers may form solid precipitates upon storage. Observe those buffers.
If a precipitate is present, place the buffers in a tube rack in the 56

C heat block and allow the tube to warm until the precipitates have dissolved fully.
9.
Label 6 eppendorf tubes as listed below. Also put your group number on each tube. a.
95% ethanol b.
ATL c.
AE1 d.
Premix e.
AE2 f. AE3
10.
Label 3 collection tubes as listed below. Also put your group number on each tube. a.
Spin tube 1 b.
Spin tube 2 c.
Spin tube 3
2

Part B. Grind and Lyse a Mosquito
1.
Pipette 500
 l of 95% ethanol into the 95% ethanol tube.
2.
Pipette 180
 l of ATL buffer into the ATL tube.
3.
Bring a sterile forceps and the 95% ethanol tube and go to the -20 Freezer A in Room 109.
4.
In the freezer, find the eppendorf tube of mosquitoes in the box labeled with your group’s number. Your instructor will tell you which species of mosquito you have.
5.
Quickly, use the sterile forceps to remove one mosquito and place it into the 95% ethanol tube. DO NOT FREEZE THAW mosquitoes repeatedly!!! Work quickly when removing the mosquito from the tube to preserve the rest for future work.
6.
Return to your lab bench with your mosquito.
7.
Use the 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.
9.
Hold the mosquito over the rinse beaker and use sterile milliQ water to thoroughly rinse the
95% ethanol off of the mosquito.
10.
Add the rinsed wingless mosquito to the ATL tube.
11.
In the ATL tube, use a pestle to grind the insect thoroughly.
12.
Pipette 20
 l proteinase K into the ATL tube.
13.
Mix the contents of the ATL tube thoroughly by pulse-vortexing for 5 – 10s.
14.
Place the ATL tube in the 56

C heat block.
15.
Incubate the ATL tube for 1 hour. If needed, you may incubate the ATL tube up to 3 hours or overnight. If an overnight incubation is chosen, the remainder of this procedure MUST be completed the next day .
16.
If the incubation is not overnight, set a timer for the desired incubation length.
17.
During the incubation: a.
As specified in the Laboratory Notebook Preparation handout, setup your lab notebook for Experiment 2. Use a glue stick to glue this protocol into your lab notebook. b.
Pipette 250
 l of AE buffer into the AE1 tube. c.
Place the AE1 tube in the 56

C heat block. d.
Pipette 200
 l of AL buffer and 200
 l of 100% ethanol (can be from 96-100% ethanol) into the premix tube. e.
Mix the contents of the premix tube thoroughly by pulse-vortexing for 5 – 10s. f.
Place a DNeasy mini spin column into spin tube 1.
3

Part C. Extract the DNA
1.
After the designated incubation time, remove the ATL tube from the 56

C heat block.
2.
Remix the contents of the premix tube thoroughly by pulse-vortexing for 5 – 10s.
3.
Pipette 400
 l of premix into the ATL tube and quickly mix the contents thoroughly 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.
4.
Pipette the mixture in the ATL tube, including any precipitate, into the DNeasy Mini spin column in spin tube 1. Make your best estimate as to what volume to set a pipette for and make sure you get as much of the mixture transferred as possible.
5.
Centrifuge spin tube 1 at 8000rpm for 1 minute.
6.
Remove the DNeasy Mini spin column from spin tube 1 and place it into spin tube 2.
7.
Pipette 500
 l of Buffer AW1 into the DNeasy Mini spin column in spin tube 2.
8.
Centrifruge spin tube 2 at 8000rpm for 1 minute.
9.
Remove the DNeasy Mini spin column from spin tube 2 and place it into spin tube 3.
10.
Pipette 500
 l of Buffer AW2 into the DNeasy Mini spin column in spin tube 3.
11.
Centrifruge spin tube 3 at 14000rpm for 3 minutes.
12.
Carefully remove the DNeasy Mini spin column from spin tube 3 so that it does NOT come into contact with any fluid and place it into the AE2 tube. a.
If the DNeasy Mini spin column does come into contact with fluid in spin tube 3, repeat the last spin.
13.
Remove the AE1 tube from the 56

C heat block.
14.
Pipette 100
 l of AE buffer from the AE1 tube directly onto the DNeasy Mini spin column in the AE2 tube.
15.
Incubate the AE2 tube at room temperature for 1 minute.
16.
Centrifuge the AE2 tube at 8000rpm for 1 minute.
17.
Remove the DNeasy Mini spin column from the AE2 tube and place it into the AE3 tube.
18.
Place the AE2 tube on ice.
19.
Pipette 100
 l of the AE buffer from the AE1tube directly onto the DNeasy Mini spin column in the AE3 tube. Place the AE1 tube on ice.
20.
Incubate the AE3 tube at room temperature for 1 minute.
21.
Centrifuge the AE3 tube at 8000rpm for 1 minute.
22.
Remove the DNeasy Mini spin column from the AE3 tube.
23.
Place the AE3 tube on ice.
24.
Discard the DNeasy mini spin column and all tubes except for the AE1, AE2, and AE3 tubes. Keep those three tubes on ice.
4

Part D: Quantify Extracted DNA
1.
Quantify the concentration of DNA in the AE2 and AE3 tubes as follows: a.
Bring a P20 pipette, tips, used tips beaker, and your AE1, AE2, and AE3 tubes on ice to your instructor, and your instructor will take you to the NanoDrop. b.
Your instructor will run the software for the NanoDrop. c.
Raise the sampling arm and pipette 1
 l of AE1 onto the lower measurement pedestal as depicted in the figure below. d.
Lower the sampling arm. Your instructor will blank the NanoDrop and will explain what a blank is. e.
When the measurement is complete, raise the sampling arm and wipe the sample from both the lower and upper pedestals using a kimwipe as depicted in the figures below.
Simple wiping prevents contamination between samples. f. Repeat steps c. – f. for the AE2 sample. g.
Repeat steps c. – f. for the AE3 sample. h.
Your instructor will print out your results and explain what they are. i. Interpret your NanoDrop results based on the information in Part E.
5

Part E: Interpreting NanoDrop Results

An absorbance spectra will be generated by the nanodrop, using 220-350 nm light. o The absorbance maximum for nucleic acids is 260nm, so the absorbance at 260nm indicates the amount of nucleic acids. o An absorbance maximum for proteins is 280nm, so the absorbance at 280nm indicates the amount of protein. o The ratio of absorbance values at 260 and 280 nm (i.e. AKA the 260/280 ratio) should be between about 1.5-1.8 for clean DNA. o For the Qiagen kit used here, the peak at about < 220 nm is a contaminate from one of the buffers in the kit (i.e. guanidinium isothiocyanate or GIT; see Figures 1-3).

As a general rule, the peak at 260nm should be large and obvious in comparison to the peak at < 220nm.

Generally speaking if the peak at 260nm is small and the peak at < 220 nm is large, you will have to use a larger volume of the DNA sample to get enough DNA for the polymerase chain reaction (PCR) experiments you will perform with these samples.
Thus the amount of GIT in the PCR reactions will of course increase. Since, GIT tends to prevent DNA molecules from interacting with each other (which is necessary for
PCR), the extra GIT will interfere with downstream PCR experiments so much the next steps may not work at all.

One caveat- if the DNA concentration is too high the absorbance at 260nm will go beyond 2.0. Dilute the sample 1:2 (i.e. equal volumes of sample and milliQ water) and repeat the absorbance until you get a workable number. Then multiple the concentration of the diluted sample by two. One can use a 1:3 or 1:4, etc dilution also if needed. Just adjust the final calculation.
6

Figure 1- A good nanodrop result- A large peak at about 260nm that is comparable in size to the peak at 220nm. The 260/280 ratio is about 2.0 indicating little to no protein contaminated the sample. This sample should be used for experiments and will probably work. The DNA concentration is 129.5 ng/
 l= 129.5
 g/ml
7

Figure 2- A mediocre nanodrop result- A peak at about 260nm that is obvious, so there is
DNA in the sample. But the peak at 260nm is not comparable in size to the peak at 220nm.
Also, there is a shoulder on the peak at 260nm at about 280nm indicating protein in the sample and the
260/280 ratio is 1.90 suggesting that it is probably clean enough to use in experiments. However, because the peak at 260nm is not distinct from the 280 nm peak, it is not clear if the estimated concentration of DNA in the sample is influenced by the protein contamination. It is possible that the concentration is less than the concentration calculated by the computer (i.e. 26.8 ng/
 l=
26.8
 g/ml). This sample probably can be used for PCR but may or may not work.
8

Figure 3- A poor nanodrop result- A peak at about 260nm missing altogether indicating little to no DNA in the sample.
Sometime a shoulder at 260nm can be seen. The small peak at >80nm is probably proteins and other contaminants in the sample. This sample will probably not work for PCR, and it should not be attempted.
9

Part F: Store Your Samples
1.
Consult with your instructor about the nanodrop results and the quality of your DNA prep.
If the DNA prep is good, it should be divided (alliquotted) into smaller volumes to avoid repeated freeze thaws of each DNA prep.
2.
Label 20 microcentrifuge tubes on the side and on the top with the information below and put the tubes on ice.
 group number/mosquito genus and species/experiment number-AE#-DNA aliquot number/date of the experiment as depicted below:
Group 1
C. melanura
#2-AE2-1
09/31/12
3.
Pipette 20
 l from the AE2 into each of the correctly labeled ten tubes. Repeat for the AE3 sample. Keep the samples on ice while pipetting.
4.
Place the samples into your group’s box in the -20 Freezer (A).
5.
WHEN YOU RECORD YOUR EXPERIMENTS IN YOUR LAB NOTEBOOK BE SURE
TO ALSO RECORD THE ABOVE INFORMATION. This way you will know exactly what tube you used for the PCR work.
10