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| __TOC__ | | __TOC__ |
| ==27 September 2010 - Ordered Primers== | | |
| * Ordered first set of primers
| | |
| * Scun2 and Scun22 (highly variable simple motifs)
| | ==CNTI Acos5== |
| ** 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)
| | |
| * [[Image:Luckau_Primer20100927.jpg|500 px]] | | ===PCR=== |
| * Try all four combinations
| | * [[Image:20120110_PCRa.png|800 px]] |
| # F - R
| | |
| # F - R pigtail
| | |
| # F M13 - R
| | ===Gel=== |
| # F M13 - R pigtail
| | |
| * If they don't hinder efforts or change optimization decisions, then may make optimization of multiplexes cheaper!
| | {| {{table}} style="text-align: center" |
| ==11 October 2010 - PCR Thermalcycler Testing==
| | |- |
| ===Purpose=== | | ! Pour !! Load !! Run |
| * 10μL in each well of full-skirt PCR plate, PCR seal
| | |- |
| * First set-up of thermalcycler, established User, Protocols, Plates and Masters
| | | 270 mL 1x TAE + 5.4 g agarose || 2 µL gel load dye + 4 µL PCR product || 160 V |
| * Data: 20101011_TKL_CyclerTest.tad
| | |- |
| ===Results===
| | | 27 µL GelRed || 6 µL ladder || 45 minutes |
| * Evaporation all around edges
| | |} |
| * Try using silicone mat?
| | |
| ===Purpose===
| | |
| * 10μL in each well of full-skirt PCR plate, PCR seal, 1 silicone mat
| | [[Image:20120110_Gela.png|800 px]] |
| * Data: 20101011_TKL_CyclerTest1Mat.tad
| | |
| ===Results===
| | * mispriming everywhere - primer pair not usable for CNHY |
| * Still evaporation around edges, but better
| | * lots of mispriming, but may be able to use conditions indicated by orange face ([[Image:orangefrownyface.png|20 px]]), if needed, under stringent conditions |
| * Try using 2 silicone mats?
| | * favorable conditions indicated by [[Image:greenhappyface.png|25 px]] |
| ==12 October 2010 - PCR Thermalcycler Testing==
| | |
| ===Purpose===
| | |
| * 10μL in each well of full-skirt PCR plate, PCR seal, 2 silicone mats
| | ===Fragment Analysis Submission=== |
| * Data: 20101012_TKL_CyclerTest2Mat.tad
| | |
| ===Results===
| | |
| * No evaporation!
| | :: [[Image:20130226_Frag.png|900 px]] |
| * 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?
| | * FedEx Tracking# [http://www.fedex.com/Tracking?language=english&cntry_code=us&tracknumbers=801124151778 801124151778] |
| ==18 October 2010 - PCR Thermalcycler Testing==
| | :: [[Image:20130226_FedEx.png|700 px]] |
| ===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
| | * UAGC Submission# PPTX |
| ===Results===
| | |
| * H6, H7 evaporated, but I think it's workable
| | |
| * Plan to do first optimization PCR tomorrow!
| | * FedEx picked up 2:44pm, 26 February |
| ==18 October 2010 - Primer Rehydration, Scun2==
| | * FedEx delivered 9:29am, 27 February |
| ===Purpose===
| | * UAGC received 10:58pm, 27 February |
| * Rehydrate Scun2 primers for testing tomorrow
| | * UAGC completed 10:41am, 28 February |
| # 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
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| * Scun2-F-M13F(-21)
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| ** 26.6nmol + 133µL Low TE = 200µM STK
| |
| * Scun2-R-pigtail
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| ** 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==
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| ===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
| |
| * (C<sub>1</sub>) (V<sub>1</sub>) = (C<sub>2</sub>) (V<sub>2</sub>)
| |
| *: (2.5mM) (5000µL) = (100mM) (V<sub>2</sub>) | |
| *: V<sub>2</sub> = 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==
| |
| * [[Image:Luckau_PCR20101019.jpg|700 px]]
| |
| * DNA = SCOC CPN 82
| |
| * 2 mats with lab tape, no evaporation!
| |
| ==21 October 2010 - Gel Scun2 F-R==
| |
| ===Make 1x TAE===
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| * 20mL 50x TAE + 980 mL NanoPure H<sub>2</sub>O | |
| ===Cast Gel===
| |
| * 100mL 1x TAE + 1.5g agarose
| |
| * large stirbar, hotplate set at 150 for about 30 min
| |
| * let cool about 20 min
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| * not enough volume! FAIL!
| |
| ===Next Try===
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| * 270mL TAE + 4.05g agarose
| |
| * (for future reference, total volume ≈ volume of gel box in cm<sup>3</sup>, ie 27cm long x 20 cm wide x 0.5 cm thick gel = 270mL of TAE)
| |
| ==25 October 2010 - Gel Scun2 F-R==
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| ===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
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| * add 27µL GelRed
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| * let cool in dark for about 30 min | |
| * pour into caster, set 2 24-tooth combs, cover with cardboard
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| * let cool in dark (had to do office hours, sat for about 2 hours) | |
| ==25 October 2010 - Ladder Mix==
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| ===Manufacturer specifications===
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| * 0.1µg ladder / mm width of well | |
| * STK at 1.0µg/µL | |
| ===Other Knowns===
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| * well width (24-tooth comb) = 6mm
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| * 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
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| * Per well: 0.6µL ladder + 4.4µL H<sub>2</sub>O + 1.5µL loading dye = 6.5µL total ladder mix
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| * (loading dye = xylene cyanol and bromophenol, made by Bohonak lab)
| |
| ===Make Ladder Mix===
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| * 120µL ladder + 880µL H<sub>2</sub>O + 300µL loading dye = 1300µL ladder mix
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