27 September 2010 - Ordered Primers
- Ordered first set of primers
- Scun2 and Scun22 (highly variable simple motifs)
- Lance et al (2009) Conservation Genetics Resources, Sceloporus undulatus
- Forward and reverse as given in publication, also reverse with pigtail (GTTTCTT) at 5' end, also forward with M13F(-21) tag (TGTAAAACGACGGCCAGT)
- Try all four combinations
- F - R
- F - R pigtail
- F M13 - R
- F M13 - R pigtail
- If they don't hinder efforts or change optimization decisions, then may make optimization of multiplexes cheaper!
11 October 2010 - PCR Thermalcycler Testing
- 10μL in each well of full-skirt PCR plate, PCR seal
- First set-up of thermalcycler, established User, Protocols, Plates and Masters
- Data: 20101011_TKL_CyclerTest.tad
- Evaporation all around edges
- Try using silicone mat?
- 10μL in each well of full-skirt PCR plate, PCR seal, 1 silicone mat
- Data: 20101011_TKL_CyclerTest1Mat.tad
- Still evaporation around edges, but better
- Try using 2 silicone mats?
12 October 2010 - PCR Thermalcycler Testing
- 10μL in each well of full-skirt PCR plate, PCR seal, 2 silicone mats
- Data: 20101012_TKL_CyclerTest2Mat.tad
- No evaporation!
- Silicone mats got stuck on lid
- Try using 2 silicone mats with tape?
- Try getting samples of different brand of silicone mats, different shape, no holes?
18 October 2010 - PCR Thermalcycler Testing
- 10µL in each well of full-skirt PCR plate, PCR seal, 2 silicone mats, lab tape on opposite sides
- Data: 20102018_TKL_CyclerTest2MatTape.tad
- H6, H7 evaporated, but I think it's workable
- Plan to do first optimization PCR tomorrow!
==18 October 2010 - Primer Rehydration, Scun2
- Rehydrate Scun2 primers for testing tomorrow