- Use the cycling conditions labeled as M13 (under Lab or Martin) on Hulcr lab thermocycler.
- For extra care (ie publications), prepare a replicate PCR for each extraction.
Running the gel
- Prepare a 1% agarose gel (careful, it will be fragile). Make sure the gel is level when pouring!
- Use extra reagents, but make sure the volume will fit in the comb you use for making the wells:
- When loading, arrange samples from similar morphotypes together. If replicate PCRs were prepared, do not group them directly next to corresponding replicate sample.
- Place both a 100bp and 1000 bp ladder on either side of every 5-10 reactions ran. This will aid in reading the gel, and will help to show if the DNA is moving at different speeds in different parts of the gel.
- Run for up to 3 hours at 100-110V. Check every 45min-1 hour and take photo. The longer you run the gel, the more differentiated the bands will become, making it easier to read.
Reading the gel:
- Use a photo editor (ie photoshop) to adjust the contrast/brightness so that all bands can be seen and differentiated between samples.
- Use a line tool in the photo editor to connect bands in ladders with the same lengths. Use the slope of these lines to gage the distances traveled between samples.
- Note any differences in fragment location and presence/absence between samples. Same species should be basically identical in their arrangement. Samples that appear different should be sequenced at a variable locus for confirmation.