Protocol by Helen Yang, transcribed by Janki Patel.

Drosophila genetics allows us to phenotypically score for the presence of transgenes in our fly stocks. There may come a time when you make a mistake (just how I did which lead me to write this page) and you need to check for the transgene using PCR. Alternatively, you may also need to run PCR when you are trying to recombine two transgenes onto the same chromosome.

Because this protocol uses a roughly digested fly as the PCR template, it doesn’t work well for PCR products of more than a few kb. If you’re just testing for the presence/absence of a transgene and have flexibility in the product size, try to target ~300-700 bp.

Reagents you need:

Squishing buffer (SQIB) (for extracting DNA)

Proteinase K (digest proteins)

PCR primers, from IDT (starting point of DNA synthesis)

5X taq buffer (pH control, Mg2+ ions - generally provides a chemical environment for the reaction)

GoTaq (DNA polymerase for DNA synthesis)

dNTPs (the building blocks for the DNA product)

miliqH2O

PCR tubes

DNA ladder

Ethidium bromide (to visualise the DNA)

Location of things

  1. dNTP aliquots: -20°C main lab
  2. 5X GoBuffer: -20°C equipment room (use the green one if you’re just running your PCR out on a gel for visualization; it contains loading dye so the PCR can be directly loaded onto a gel)
  3. GoTaq: -20°C equipment room (in the blue table-top cooler; when grabbing this, take the whole cooler, which keeps all the tubes cold on the bench for some time. Only take this out of the freezer when you’re ready to make the PCR reaction mix and put it back in the freezer as soon as you’re done adding the GoTaq)
  4. 100mM primer stock: store at -20°C
  5. 10mM primer stock: store at 4°C for up to a week or two
  6. DNA ladder: main lab 4°C
  7. Proteinase K aliquots at 20mg/ml: -80°C equipment room
  8. PCR tubes, electrophoresis equipment: back bench in a drawer labelled “PCR equipment”
  9. SQIB buffer: in 50mL conical in a tube rack on the chemical bench.

The protocol is written in the order you would perform it.

  1. Anesthetise the flies on CO2 pads and put one fly in each PCR tube. Label the tubes with numbers on the top and along the side close to the top. Make a note of the associated genotype in your notebook. Once you have all the flies in tubes, put them in the -20°C for ~10 minutes.
  2. Now, make the squishing buffer. You need 50µL per fly (49µL SQIB buffer + 1µL proteinase K (20mg/mL, from NEB)). We have SQIB ready to use on the shelf, just add proteinase K to it. SQIB buffer is 10mM Tris pH 8.3 (from 0.1M stock) + 1mM EDTA pH 8 (from 0.5M stock) + 25mM NaCl (from 1M stock). The Proteinase K aliquots are in the -80.
  3. Aspirate 50uL into your pipet tip and crush the fly in the tube, then eject the liquid. (flies are surprisingly hydrophobic and crushing before ejecting the liquid saves you from having to chase a floating fly)
  4. When you have all your PCR tubes ready with squished flies in the buffer, run the following reaction in the thermocycler (Use the SQIB protocol in the thermocycler). This fly digest is stable for ~1 day at 4°C but it’s much better to proceed directly to the PCR. Only use flies that aren’t freshly digested if you have no other choice (e.g. the PCR failed and you need to check those specific individuals)
  5. 37°C 30 mins
  6. 85°C 2 mins (to denature proteinase K)
  7. 4°C for holding
  8. While the SQIB protocol is running, make the PCR reaction mix. If you need to reconstitute your primers, add appropriate amount of TE buffer to the tube to make 100mM stock solution. Do not pipet up and down or vortex, simply flick the tube a couple times and watch as the “salt” residue disappear from the bottom of the tube. You now have a 100µM primer solution. Store this at -20°C. Then make a 10µM working stock in water. You can leave the 10µM stock at 4°C for a week or two. The 100µM stock is in TE because the primers remain more stable in TE buffer. Make PCR reaction mix of everything but the fly template. Make enough for the number of samples you have + 1. Then pipette the appropriate amount of PCR reaction mix into PCR tubes (24µL per tube). When the SQIB protocol is done running, add 1µL of the supernatant as a template for each reaction to the PCR tubes already containing the PCR reaction mix (with the appropriate combination of which fly and which primers for your desired PCRs). Avoid adding fly bits to the PCR reaction. Each PCR reaction contains (total 25µL volume):
  9. 5µL 5X GoTaq Buffer
  10. 0.5µL dNTPs (10mM, from NEB)
  11. 1.25µL primer 1 (10µM)
  12. 1.25µL primer 2 (10µM)
  13. 0.25µL GoTaq (keep this in the blue cooler)
  14. 1µL template (from digested fly; exclude from the PCR reaction mix)
  15. 15.75µL H2O
  16. Run the TRiP program (or make your own) on the thermocycler to perform the PCR reaction:
  17. 95°C 3mins
  18. 95°C 30sec —–
  19. 57°C 30sec | cycle 35 times
  20. 72°C 30sec —– (depends on your product length: 1 minute per 1kb, but less than 30 sec is not recommended)
  21. 72°C 7 mins
  22. hold at 4°C
  23. While the reaction is running, make 2.5% agarose gel. Add 2.5g of agarose in 100mL of TAE buffer. Zap the solution for 30 seconds at a time in the microwave. Once the agarose is in solution, add 1µL of Ethidium bromide (1:100,000). The ethidium bromide is to visualise the DNA under UV illumination.
  24. Set the combs in place and firmly place the gel holder in to secure the edges with the rubber gasket and pour in the gel. Let it set while you wait for the PCR reaction to run.
  25. Fill the electrophoresis unit with TEA until the designated line. Add 10-15µL of the 1kb plus DNA ladder from invitrogen and add 10µL of the PCR reaction mix into the well. note: we have two kinds of GoBuffers, one has the loading dye already in it and one doesn’t. If you are using the one that does then you do not need to add loading dye to your PCR reaction mix before loading it into the gel.
  26. Run the gel for 30 minutes at 120V - 150V. This time will depend on the size of your product. If you’re using the green GoBuffer, the yellow dye runs below 100 bp.
  27. Visualise the gel.

Some comments:

  1. If it is your first time testing a PCR primer, run a control primer to ensure the reaction worked.
  2. Include a positive and a negative control fly for the primers.
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