TITLE: Ethanol extraction protocol
AUTHORS: Nannie 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Developed for IBA at the NRM DNA lab.</div></div>

[STEPS]
?. Decant the ethanol through a mesh and add new ethanol to the insects.
?. Filter the ethanol: method 2.1 or 2.2
?. Vacuum pumpPut the Sterivex filters on the vacuum pump with the large syringe attached to the filter. Turn on the pump and pour the ethanol into the syringe.The filtered ethanol will not be saved.
?. ManuallyFilter the ethanol manually through the large syringe.The filtered ethanol can be saved.
?. Add 540 µl Buffer ATL from the extraction kit to the filter.Add 60 µl Proteinase K from the extraction kit to the filter.Put the filters in a shaking incubator overnight at 56°C.Remember to screw the caps on properly, otherwise the buffer will evaporate!
?. Extract the DNARemove the buffer from the filter using the small syringe and put the liquid in a 1.5 ml Eppendorf tube.
?. Qiagen Blood and Tissue kit protocol:Vortex 15 seconds.Add 200 µl Buffer AL. Mix thoroughly by vortexing.Incubate at 56°C for 10 min.Add 200 µl ethanol (96-100%). Mix thoroughly by vortexing.Pipet the mixture into a DNeasy Mini spin column placed in a 2 ml collection tube. Centrifuge at ≥6000 x g (8000 rpm) for 1 min. Discard the flow-through and collection tube.Place the spin column into a new collection tube. Add 500 µl Buffer AW1. Centrifuge for 1 min. at 8000 rpm. Discard the flow-through and collection tube.Place the spin column into a new collection tube. Add 500 µl Buffer AW2. Centrifuge for 3 min. at 20,000 x g (14,000 rpm). Discard the flow-through and collection tube.Transfer the spin column to a new 1.5 ml Eppendorf tube.Elute the DNA by adding 100 µl Buffer AE to the center of the column membrane. Incubate for 1 min. at room temperature (15-25°C). Centrifuge for 1 min. at 8000 rpm. Store the extracted DNA in freezer.
?. PCR for COIPrimers F:COIBF3_P5_ins1, COIBF3_P5b_ins2, COIBF3_P5b_ins3, COIBF3_P5ACACTCTTTCYCCHCGPrimers R:COIBR2_P7_sub1, COIBR2_P7_ins1, COIBR2_P7_ins2, COIBR2_P7GRGACTGGAGRAAYCADilute and mix primers to 10 µM (stock is 100 µM).
?. PCR mix for 20 µl total volume/sample:2X Qiagen Master Mix10 µl10 µM primer mix F2 µl10 µM primer mix R2 µlddH2O2 µlDNA template4 µl
2X Qiagen Master Mix10 µl10 µM primer mix F2 µl10 µM primer mix R2 µlddH2O2 µlDNA template4 µl
?. Electrophoresis
?. Mix a 1% agarose gel (buffer, agarose, gelgreen).Remember to put a black box on top to keep it from the light.Dilute Blue loading dye 1:2 ddH2O.Use 1 µl Ladder 100 bp (per row).Mix 1 µl loading dye with 4 µl PCR product and add to wells.Run at 80-90 V.Visualize the gel on the UV plate and take a picture with the camera.
?. PCR program  Step    Temp [°<b style="mso-bidi-font-weight:
  normal">C]    Time [s]    Number of cycles    Taq activation    95    900    1    Denaturation    94    30    35    Annealing    50    90    35    Extension    72    90    35    Final extension    72    600    1    End    8    forever
Step    Temp [°<b style="mso-bidi-font-weight:
  normal">C]    Time [s]    Number of cycles    Taq activation    95    900    1    Denaturation    94    30    35    Annealing    50    90    35    Extension    72    90    35    Final extension    72    600    1    End    8    forever