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
- 28.3nmol + 141.5 Low TE = 200µM STK
- 29.2nmol + 146µL Low TE = 200µM STK
- 26.6nmol + 133µL Low TE = 200µM STK
- 30.5nmol + 152.5µL Low TE = 200µM STK
- stored overnight in fridge to rehydrate completely before making dilution to 20µM
18 October 2010 - dNTP Mix
- Mix dNTPs and aliquot for PCR
- Have: Invitrogen 100mM dNTP Set (catalog# 10297-018) (250µL of 25µmol in H2O @ 100mM each)
- Want: 10mM dNTP mix (2.5mM each), 5 aliquots of 1000µL
- V2=125µL of each dNTP
- 125µL dATP + 125µL dTTP + 125µL dCTP + 125µL dGTP + 4500µL H2O = 5000µL dNTP Mix @ 10mM (2.5mM each dNTP)
- Used NanoPure H20 from room 325
- Aliquots: 1000µL into each of 5 snap-caps; 4 stored in freezer, 1 stored in fridge for immediate use
19 October 2010 - PCR Scun2 F-R
- DNA = SCOC CPN 82
- 2 mats with lab tape, no evaporation!
21 October 2010 - Gel Scun2 F-R
Make 1x TAE
- 20mL 50x TAE + 980 mL NanoPure H2O
- 100mL 1x TAE + 1.5g agarose
- large stirbar, hotplate set at 150 for about 30 min
- let cool about 20 min
- not enough volume! FAIL!
- 270mL TAE + 4.05g agarose
- (for future reference, total volume ≈ volume of gel box in cm3, ie 27cm long x 20 cm wide x 0.5 cm thick gel = 270mL of TAE)
25 October 2010 - Gel Scun2 F-R
- 270mL TAE + 4.05g agarose in 500mL Erlenmyer flask
- large stirbar, hotplate set at 150, stir set at 15 for about 45 min, until clear
- add 27µL GelRed
- let cool in dark for about 30 min
- pour into caster, set 2 24-tooth combs, cover with cardboard
- let cool in dark (had to do office hours, sat for about 2 hours)
25 October 2010 - Ladder Mix
- 0.1µg ladder / mm width of well (STK at 1.0µg/µL)
- well width (24-tooth comb) = 6mm
- 6mm x 0.1µg ladder