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
Purpose
- 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
Results
- Evaporation all around edges
- Try using silicone mat?
Purpose
- 10μL in each well of full-skirt PCR plate, PCR seal, 1 silicone mat
- Data: 20101011_TKL_CyclerTest1Mat.tad
Results
- Still evaporation around edges, but better
- Try using 2 silicone mats?
12 October 2010 - PCR Thermalcycler Testing
Purpose
- 10μL in each well of full-skirt PCR plate, PCR seal, 2 silicone mats
- Data: 20101012_TKL_CyclerTest2Mat.tad
Results
- 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
Purpose
- 10µL in each well of full-skirt PCR plate, PCR seal, 2 silicone mats, lab tape on opposite sides
- Data: 20102018_TKL_CyclerTest2MatTape.tad
Results
- H6, H7 evaporated, but I think it's workable
- Plan to do first optimization PCR tomorrow!
18 October 2010 - Primer Rehydration, Scun2
Purpose
- Rehydrate Scun2 primers for testing tomorrow
- Scun2-F
- Scun2-R
- Scun2-F-M13F(-21)
- Scun2-R-pigtail
Calculations
- Scun2-F
- 28.3nmol + 141.5 Low TE = 200µM STK
- Scun2-R
- 29.2nmol + 146µL Low TE = 200µM STK
- Scun2-F-M13F(-21)
- 26.6nmol + 133µL Low TE = 200µM STK
- Scun2-R-pigtail
- 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
Purpose
- Mix dNTPs and aliquot for PCR
Calculations
- 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
- (C1) (V1) = (C2) (V2)
- (2.5mM) (5000µL) = (100mM) (V2)
- V2 = 125µL of each dNTP
Wet Work
- 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
Cast Gel
- 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!
Next Try
- 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 - Ladder Mix
Manufacturer specifications
Other Knowns
- well width (24-tooth comb) = 6mm
- Jeremy (Bohonak lab) uses 5µL PCR product + 1.5µL dye
Calculations
- 6mm x (0.1µg ladder)/(1mm width) x (1µL/1µg) = 0.6µL ladder per well
- Per well: 0.6µL ladder + 4.4µL H2O + 1.5µL loading dye = 6.5µL total ladder mix
- (loading dye = xylene cyanol and bromophenol, made by Bohonak lab)
Make Ladder Mix
- 120µL ladder + 880µL H2O + 300µL loading dye = 1300µL ladder mix
25 October 2010 - Gel Scun2 F-R
Cast Gel
- 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)
Load Gel
Run Gel
- use top volt-box
- Power On
- DC On
- Voltmeter: push to right
- Adjust red knob to 90-120V (using red scale of left-side gauge)
- 90V 3p-4:30p
- 120V 4:30p-4:45p
Image Gel
- nothing...
 
What Happened?
- UV light?
- should be working, when turned off, image black, when turned on, can see gel (but only agarose gel)
- Gel Stain?
- used as according to Clark's protocol (10% volume of 3x Gel Red)
- check product protocol
- Next:
- try a bunch of different trials of ladder, gel stain, on small gel
26 October 2010 - GelRed info
26 October 2010 - Gel Test
Cast Gel
- small caster: 10cm x 10cm x 0.5cm = 50mL TAE
- 50mL TAE + 1g agarose in 250mL Erlenmyer flask
- small stirbar, hotplate set at 200, stir set at 150 for about 10 min, until clear
- let cool (about 5 min)
- add 5µL GelRed 10,000X, swirl
- pour into caster, set comb
- let set (about 5min)
Load Gel
- 4µL Tara Ladder (T)
- 4µL Justin Ladder (J)
- 2µL Sample (H6) (added minute after applying current)
Run Gel
- 110V, 20 min
- 150V, 10 min
Image Gel
- Ladders show!
- both Tara's and Justin's
 - Sample is very faint, may be due to smaller volume placed
- ~200-300bp fragment
- (Lance et al 2009, says Scun2 has amplicon length of 131-191)
 - do another gel with subset of PCR product, full volume
28 October 2010 - Gel Scun2 F-R
Cast Gel
- medium caster: 20cm x 13cm x 0.5cm = 130mL TAE
- 130mL TAE + 2.6g (2%) agarose in 250mL Erlenmyer flask
- small stirbar, hotplate set at 200, stir set at 150, for about 15min, until clear
- let cool (about 5 min)
- add 13µL GelRed 10,000X, swirl
- pour into caster, set 36-tooth comb
- let set (about 10min)
Load Gel
- 4µL Ladder
- 4µL Sample w/ loading dye
Run Gel
Image Gel
- Nada, zip, zilch ... poopy
   - WTF?
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Tara's Lab Notebook
