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

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==27 September 2010 - Ordered Primers==
==27 September 2010 - Ordered Primers==
* Ordered first set of primers
* Ordered first set of primers
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* Have: Invitrogen 100mM dNTP Set (catalog# 10297-018) (250µL of 25µmol in H2O @ 100mM each)
* 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
* Want: 10mM dNTP mix (2.5mM each), 5 aliquots of 1000µL
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* (C1)(V1)=(C2)(V2)
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* (C<sub>1</sub>) (V<sub>1</sub>) = (C<sub>2</sub>) (V<sub>2</sub>)
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*: (2.5mM)(5000µL)=(100mM)(V2)
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*: (2.5mM) (5000µL) = (100mM) (V<sub>2</sub>)
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*: V2=125µL of each dNTP
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*: V<sub>2</sub> = 125µL of each dNTP
===Wet Work===
===Wet Work===
* 125µL dATP + 125µL dTTP + 125µL dCTP + 125µL dGTP + 4500µL H2O = 5000µL dNTP Mix @ 10mM (2.5mM each dNTP)
* 125µL dATP + 125µL dTTP + 125µL dCTP + 125µL dGTP + 4500µL H2O = 5000µL dNTP Mix @ 10mM (2.5mM each dNTP)
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===Calculations===
===Calculations===
* well width (24-tooth comb) = 6mm
* well width (24-tooth comb) = 6mm
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* 6mm x 0.1µg ladder
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* 6mm x $0.1µg ladder \over 1mm width$ x $µL \over 1.0µg$ = 0.6µL ladder per well
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* from Jeremy (Bohonak lab): uses 5µL PCR product + 1.5µL dye
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* Per well: 0.6µL ladder + 4.4µL H<sub>2</sub>O = 5µL ladder mix
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* (xylene and bromophenol blue)

Revision as of 17:00, 25 October 2010

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 - 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)

25 October 2010 - Ladder Mix

Manufacturer specifications

  • 0.1µg ladder / mm width of well (STK at 1.0µg/µL)

Calculations

  • well width (24-tooth comb) = 6mm
  • 6mm x $0.1µg ladder \over 1mm width$ x $µL \over 1.0µg$ = 0.6µL ladder per well
  • from Jeremy (Bohonak lab): uses 5µL PCR product + 1.5µL dye
  • Per well: 0.6µL ladder + 4.4µL H2O = 5µL ladder mix
  • (xylene and bromophenol blue)



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