Drosophila 


  

Drosophila Gel

Figure 1. Annealing temperature optimisation experiment using Drosophila melanogaster DNA extracted with prepGEM Insect. Primers used targeted the GAPDH gene. Forward:  Reverse: PCR was performed using AmpliTaq DNA polymerase (Life Tech).


Notes

 

1. For dried insects:

• Break up the exoskeleton with forceps

• Pre-soak overnight at 4°C in 40 µl of 1x BLACK buffer.

• Add 1 µl of prepGEM and proceed to Step 3.


2. For very small insects, volumes can be reduced.


3. There will be little apparent reduction in the amount of visible material. The purpose of the step is to lyse the cells in order to extract DNA and to strip the DNA of nucleoproteins. Excessive digestion will release more potential inhibitors into the solution. 5 minutes is sufficient in most cases.

 
4. Centrifugation should not be necessary but if you want to remove sediment, use a gentle spin (5000 x g for 2 min). Harder spins will sediment the high molecular weight DNA produced by the ZyGEM kits.


Downloadable PDF files

 

• ZyGEM method optimisation (PDF 799k)

• DNA extraction from insects (PDF 669k)

• prepGEM Insect QuickStart Guide (PDF 714k)

• DNA quant using fluorescent dyes (PDF 556k)

• prepGEM MSDS (PDF 181k)

 

This method is recommended for ZyGEM DNA extraction directly from insect tissue.  Incubations can be performed either in a thermal cycler, water bath, or using an automated robotic workstation fitted with Peltier temperature-controlled heating blocks.


Work area Preparation

All manipulations should be performed in a clean room or a PCR hood. Use only certified DNA-free tubes and reagents and wash surfaces likely to come into contact with the samples with 0.05% hypochlorite bleach. Rinse thoroughly with DNA-free water.

Insect workflow

Extraction Method

Approximately 1-2 mm3 of total tissue should be used. 

1. Insects or parts of insects should be placed in a thin-walled PCR tube for extraction. for some insects, better results are obtained by grinding the insect with the pipette tip.

2. Add to the material:

  • 35 µl of PCR-grade water
  • 4 µl of 10x buffer BLACK
  • 1 ul prepGEM™

3. Incubate at 75 °C for 15 minutes.
4. Incubate at 95°C for 5 minutes.
5. Centrifuge 2 minutes at 5,000 x g if required.

NOTE: Centrifugation at high speeds can sediment gDNA.

Typically, the supernatant from fresh insects should be diluted 1:10 in water and 1 µl used in a 25 µl PCR. For older or degraded samples, undiluted extracts can be used and the volume increased.


Technical Tips

  • The prepGEM™ Insect procedure can be automated using most liquid handling robots.
  • prepGEM™ Insect is a preparative method for DNA extraction - it is not a purification protocol.  Its purpose is to lyse cells and to strip the DNA of nucleoproteins. The formulation creates DNA that can be used for SNiPs, STRs, quantitative, multiplex and routine PCR applications.
  • prepGEM™ extracted DNA is largely single-stranded because of the heat step. If double-stranded DNA is required, the 95° step can be omitted and standard downstream purification procedures can be used.
  • For accurate yield assessment, a qPCR is recommended. DNA produced by prepGEM™ is approximately 90% single-stranded. If standard fluorescent chelating dyes are to be used for quantification, then this factor should be taken into consideration.
  • As with any preparative method for nucleic acid extraction, for best results prepare and manage samples at 4ºC, or on ice, before and after extraction.
  • When storing sample after extraction, aspirate the supernatant when cool and store separately from the tissue.
  • Depending on the type of insects present in a sample and their state of desiccation, grinding and/or re-hydration may be required to achieve optimal yield.
  • The use of EDTA in the sample-handling protocol can reduce enzyme performance in the extraction. This problem can be overcome by adding CaCl2 to a final concentration of 200 µM.
  • prepGEM™ is stable for 30 days at 4º C; for longer-term storage prepGEM™ should be stored at -20ºC.
  • For long-term storage of the extracted DNA, add TE buffer to 1x and store at -20°C.