PROTOCOLS
Phenol Chloroform DNA Extraction (Linton et al. 1999).
Single mosquito are homogenised in a 1.5 eppendorf tube using a plastic pestle
(Anachem), in 100 ml of extraction buffer containing 0.2 M sucrose, 0.1 M Tris–
HCl, 50 mM EDTA and 0.5% SDS (Samson and Wagnez, 1980). Between
successive extractions, plastic pestles are soaked in 0.25 mM HCL, to avoid
cross-contamination of DNA samples and then washed thoroughly in distilled
H2O, to get rid of any traces of HCL.
The mixture is heated at 65°C for 10 minutes in a Techno Dry-Block® DB-3A
(Genetic Research Instrumentation Ltd, England), 24 μl of 5mM KOAc (pH 9.0) is
added and the tubes are placed on ice for a further 10 minutes. Following
centrifugation at 13,200 rpm for 10 minutes, the supernatant is transferred to a
clean 1.5 ml eppendorf, then 1 μl of 20 ug / ml RNAse is added and the tubes are
transferred to a 37°C oven for 30 minutes.
Following 100 μl of PCA (Phenol/Chloroform / Isoamyl, 25:24:1) is added and the
tubes well vortexed, before centrifuging at 13.200 rpm for 1 minutes. The phenol
facilitated the precipitation of fats and lipid membranes and the chloroform acted
to break down proteins. The supernatant is removed, transferred to new
eppendorf and 100 μl of chloroform added before again vortexing and
centrifuging for 1 minute. This additional chloroform extraction ensured complete
removal of phenol from the purified DNA, which could otherwise inhibit the Taq
DNA polymerase in subsequent PCR reactions. Eighty μl of each supernatant is
transferred to a new eppendorf and eighty μl of isopropanol is added, and
samples stored at – 20 °C overnight.
Samples are removed from the freezer and centrifuged at 13.200 rpm for 10
minutes. Isopropanol is decanted and the precipitate washed with 100 μl of cold
70% ethanol and spin for 10 minutes. The ethanol is decanted and the ethanol
wash repeated with cold 100% ethanol. Ethanol is again removed, and the
samples are desiccated in a speed vacuum for 20 minutes or in the over 37° for
30 minutes. 100 μl of 10mM Tris-HCl, 0.1 mM EDTA (Ph8.0) buffer (or dH2O) is
added and the DNA left to resuspend at room temperature overnight.
Following total resuspension, the tube are carefully labelled and stored in 200 μl
PCR tubes (Greiner Labortechnik Ltd, Glos, England) at -70 °C until required.
Isolation of total DNA from Animal Tissues (Qiagen).
1. Place the abdomen in a 1.5 ml microcentrifuge tube and add 180 μl Buffer
ATL.
2. Add 20 μl proteinase K, mix by vortexing and incubate at 55°C for 3 hours.
The samples can be lysed overnight; this will not affect them adversely.
3. Vortex for 15 seconds. Add 200 μl Buffer AL to the sample, mix thoroughly
by vortexing and incubate at 70°C for 10 minutes.
4. Add 200 μl ethanol (96-100%) to the sample, and mix thoroughly by
vortexing.
5. Pipet the mixture into the DNeasy mini spon Column placed in a 2 ml
collection tube. Centrifuge at 8000 rpm for 1 min. Discard flow-trough and
collection tube.
6. Place the DNeasy mini spin column in a new 2 ml collection tube, add 500
μl Buffer AW1, and centrifuge for 1 min at 8000 rpm. Discard flow-through
and collection tube.
7. Place the DNeasy mini spin column in a 2 ml collection tube, add 500 μl
buffer AW2, and centrifuge for 3 min at 14000 rpm to dry the DNeasy
membrane. Discard the flow-through and collection tube.
8. Place the DNeasy mini spin column in a clean 1.5 ml or 2 ml
microcentrifuge tube and pipet 200 μl buffer AE directly onto the DNeasy
membrane. Incubate at room temperature for 1 min and then centrifuge for
1 min at 8000 rpm to elute.
9. Store the tubes until it will be used.
You can to decide which to use (Phenol – Chloroform or Qiagen).
Master mix for PCR reactions
Mix for one reaction using Taq Polimerase (Bioline)
25.5 μl
ddH20
5 μl
5.8 S primer a 5 uM
5 μl
28 S primer a 5 uM
5 μl
dNTPs a 2 uM
5 μl
10xNH4 Buffer
2.5 μl
25mM MgCl2
0.1 μl
Taq polymerase (BioLine)
Vortex well and for each PCR adds 2 μl DNA plus 48 μl master mix.
Note: You can to put one or two legs of the mosquito in the PCR mix directly
without DNA extraction, it works very well (48 ul of master mix pluss one or two
legs).
THERMOCYCLER CONDITIONS
ITS2 (5.8 S- 28 S)
COI (Ubc 6 – Ubc 9)
1. 94°C for 2 minutes
2. 94 °C for 30 seconds
3. 57 °C for 1 minute
35 times
4. 72 °C for 30 seconds
6. 72 °C for 10 minutes
7. 10 °C hold
Primers
ITS2 (Collins & Paskewitz, 1996)
5.8 S: ATC ACT CGG CTC GTG GAT CG
28 S: GAC TAC CCC CTA AAT TTA AGC AT
Sequence Reaction
1-2 ng DNA per 100 bps of product
1 pMol of Primer
3 μl Big Dye Dilution Buffer form Kit
1 μM big Dye Terminador Mix
ddH2O up to 10 μl
Cycle Sequence
To do a hot start without the Big Die Terminator Reaction Mix for 5 minutes at 96
°C (step 1).
The samples are then transferred to ice and the Big Dye added and mixed by
pipetting up and down 2 or 3 times. The samples are returned to the Thermal
Cycler for follow with the cycle sequences (step 2).
1. 96 °C for 5 minutes
2. 96 °C for 10 seconds
3. 50 °C for 5 seconds
25 Cycles
4. 60 °C for 4 minutes
5. Cool to 4 °C
SOLUTIONS
Grinding Buffer (Linton et al. 2001)
To make 50 ml:
5 ml Tris-HCl at pH 8.0
3.42 g sucrose
2.5 g SDS
0.86g EDTA
Fill to 50 ml with ddH20