In this exercise, you will test for changes in methylation
of the coding sequence of heat shock protein 70 in response to heat shock in Tigriopus using restriction enzyme digests with MspI (methylation insensitive) and HpaII (methylation sensitive)
DNA extraction and enzyme digest:
1) remove as much water as possible from tube containing 1 copepod. Add 30 ul of chelex, vortex, spin down, incubate at 95˚C for 20 minutes.
2) Vortex chelex solution again, spin down for 1 minute on high speed
3) Put 5ul of DNA template into each of 3 pcr tubes – one for each enzyme and one for the ‘water only’ control.
4) To each tube add: 0.5 ul enzyme (or milliQ water), 2ul of cutsmart buffer, 12 ul of water.
5) Incubate at 37˚C for 2 hours, followed by 5 min at 95˚C.
6) To each enzyme digest tube, add 0.2 ul Taq, 0.5ul Forward+Reverse HSP70 primer mix, 1.6 ul dNTPs
7) PCR cycle: “EPI”