User:Tara K. Luckau/Notebook/Team ConGen/Entry Base

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Tara's Lab Notebook Main project page

Contents

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
  1. F - R
  2. F - R pigtail
  3. F M13 - R
  4. 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
  1. Scun2-F
  2. Scun2-R
  3. Scun2-F-M13F(-21)
  4. 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



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