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

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(25 October 2010 - Ladder Mix)
(25 October 2010 - Gel Scun2 F-R)
Line 102: Line 102:
* pour into caster, set 2 24-tooth combs, cover with cardboard
* 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)
* let cool in dark (had to do office hours, sat for about 2 hours)
 +
===Load Gel===
 +
* Top:
 +
# B1
 +
# C1
 +
# D1
 +
# E1
 +
# F1
 +
# G1
 +
# H1
 +
# Ladder
 +
# A2
 +
# B2
 +
# C2
 +
# D2
 +
# E2
 +
# F2
 +
# G2
 +
# H2
 +
# A3
 +
# B3
 +
# C3
 +
# D3
 +
# E3
 +
# F3
 +
# G3
 +
# H3
 +
* Bottom:
 +
# A4
 +
# B4
 +
# C4
 +
# D4
 +
# E4
 +
# F4
 +
# G4
 +
# H4
 +
# A5
 +
# B5
 +
# C5
 +
# D5
 +
# E5
 +
# F5
 +
# G5
 +
# H5
 +
# Ladder
 +
# A6
 +
# B6
 +
# C6
 +
# D6
 +
# E6
 +
# F6
 +
# G6
 +
===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
 +
==25 October 2010 - Ladder Mix==
==25 October 2010 - Ladder Mix==
===Manufacturer specifications===
===Manufacturer specifications===

Revision as of 18:35, 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)

Load Gel

  • Top:
  1. B1
  2. C1
  3. D1
  4. E1
  5. F1
  6. G1
  7. H1
  8. Ladder
  9. A2
  10. B2
  11. C2
  12. D2
  13. E2
  14. F2
  15. G2
  16. H2
  17. A3
  18. B3
  19. C3
  20. D3
  21. E3
  22. F3
  23. G3
  24. H3
  • Bottom:
  1. A4
  2. B4
  3. C4
  4. D4
  5. E4
  6. F4
  7. G4
  8. H4
  9. A5
  10. B5
  11. C5
  12. D5
  13. E5
  14. F5
  15. G5
  16. H5
  17. Ladder
  18. A6
  19. B6
  20. C6
  21. D6
  22. E6
  23. F6
  24. G6

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

25 October 2010 - Ladder Mix

Manufacturer specifications

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

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



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