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| __TOC__ | | __TOC__ |
| ==27 September 2010 - Ordered Primers==
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| * Ordered first set of primers
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| * Scun2 and Scun22 (highly variable simple motifs)
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| ** Lance et al (2009) Conservation Genetics Resources, Sceloporus undulatus
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| * Forward and reverse as given in publication, also reverse with pigtail (GTTTCTT) at 5' end, also forward with M13F(-21) tag (TGTAAAACGACGGCCAGT)
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| * [[Image:Luckau_Primer20100927.jpg|500 px]]
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| * Try all four combinations
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| # F - R
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| # F - R pigtail
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| # F M13 - R
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| # F M13 - R pigtail
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| * If they don't hinder efforts or change optimization decisions, then may make optimization of multiplexes cheaper!
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| ==11 October 2010 - PCR Thermalcycler Testing==
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| ===Purpose===
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| * 10μL in each well of full-skirt PCR plate, PCR seal
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| * First set-up of thermalcycler, established User, Protocols, Plates and Masters
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| * Data: 20101011_TKL_CyclerTest.tad
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| ===Results===
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| * Evaporation all around edges
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| * Try using silicone mat?
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| ===Purpose===
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| * 10μL in each well of full-skirt PCR plate, PCR seal, 1 silicone mat
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| * Data: 20101011_TKL_CyclerTest1Mat.tad
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| ===Results===
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| * Still evaporation around edges, but better
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| * Try using 2 silicone mats?
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| ==12 October 2010 - PCR Thermalcycler Testing==
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| ===Purpose===
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| * 10μL in each well of full-skirt PCR plate, PCR seal, 2 silicone mats
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| * Data: 20101012_TKL_CyclerTest2Mat.tad
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| ===Results===
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| * No evaporation!
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| * Silicone mats got stuck on lid
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| * Try using 2 silicone mats with tape?
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| * Try getting samples of different brand of silicone mats, different shape, no holes?
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| ==18 October 2010 - PCR Thermalcycler Testing==
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| ===Purpose===
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| * 10µL in each well of full-skirt PCR plate, PCR seal, 2 silicone mats, lab tape on opposite sides
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| * Data: 20102018_TKL_CyclerTest2MatTape.tad
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| ===Results===
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| * H6, H7 evaporated, but I think it's workable
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| * Plan to do first optimization PCR tomorrow!
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| ==18 October 2010 - Primer Rehydration, Scun2==
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| ===Purpose===
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| * Rehydrate Scun2 primers for testing tomorrow
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| # Scun2-F
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| # Scun2-R
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| # Scun2-F-M13F(-21)
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| # Scun2-R-pigtail
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| ===Calculations===
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| * Scun2-F
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| ** 28.3nmol + 141.5 Low TE = 200µM STK
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| * Scun2-R
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| ** 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
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| * Scun2-R-pigtail
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| ** 30.5nmol + 152.5µL Low TE = 200µM STK
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| * stored overnight in fridge to rehydrate completely before making dilution to 20µM
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| ==18 October 2010 - dNTP Mix==
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| ===Purpose===
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| * Mix dNTPs and aliquot for PCR
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| ===Calculations===
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| * Have: Invitrogen 100mM dNTP Set (catalog# 10297-018) (250µL of 25µmol in H2O @ 100mM each)
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| * Want: 10mM dNTP mix (2.5mM each), 5 aliquots of 1000µL
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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) (V<sub>2</sub>)
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| *: V<sub>2</sub> = 125µL of each dNTP
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| ===Wet Work===
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| * 125µL dATP + 125µL dTTP + 125µL dCTP + 125µL dGTP + 4500µL H2O = 5000µL dNTP Mix @ 10mM (2.5mM each dNTP)
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| * Used NanoPure H20 from room 325
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| * Aliquots: 1000µL into each of 5 snap-caps; 4 stored in freezer, 1 stored in fridge for immediate use
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| ==19 October 2010 - PCR Scun2 F-R==
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| * [[Image:Luckau_PCR20101019.jpg|700 px]]
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| * DNA = SCOC CPN 82
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| * 2 mats with lab tape, no evaporation!
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| ==21 October 2010 - Gel Scun2 F-R==
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| ===Make 1x TAE===
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| * 20mL 50x TAE + 980 mL NanoPure H<sub>2</sub>O
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| ===Cast Gel===
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| * 100mL 1x TAE + 1.5g agarose
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| * large stirbar, hotplate set at 150 for about 30 min
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| * let cool about 20 min
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| * not enough volume! FAIL!
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| ===Next Try===
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| * 270mL TAE + 4.05g agarose
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| * (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)
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|
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| ==25 October 2010 - Ladder Mix==
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| ===Manufacturer specifications===
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| * 0.1µg ladder / mm width of well
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| * STK at 1.0µg/µL
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| * [[Media:Invitrogen_1KbLadderProductManual.pdf]]
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| ===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
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| ===Calculations===
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| * 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)
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| ===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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| ==25 October 2010 - Gel Scun2 F-R== | | ==CNTI Acos5== |
| ===Cast Gel===
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| * 270mL TAE + 4.05g agarose in 500mL Erlenmyer flask
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| * 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
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| * 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)
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| ===Load Gel===
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| *[[Image:BlankGelSchematicTop.jpg|600 px]]
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| ===Run Gel===
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| * use top volt-box
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| * Power On
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| *: DC On
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| *: Voltmeter: push to right
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| *: Adjust red knob to 90-120V (using red scale of left-side gauge)
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| * 90V 3p-4:30p
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| *: 120V 4:30p-4:45p
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| ===Image Gel===
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| * nothing...
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| *: [[Image:20101025_Top.TIF|300 px]] [[Image:20101025_Bottom.TIF|300 px]]
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|
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| ===What Happened?===
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| * UV light?
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| ** should be working, when turned off, image black, when turned on, can see gel (but only agarose gel)
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| * Gel Stain?
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| ** used as according to Clark's protocol (10% volume of 3x Gel Red)
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| ** check product protocol
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| * Next:
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| ** try a bunch of different trials of ladder, gel stain, on small gel
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|
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| ==26 October 2010 - GelRed info== | | ===PCR=== |
| * Yesterday, used 27µL 3x Gel Red into warm agarose/TAE mix (precast)
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| * Biotium's Product Information sheet on GelRed: [[Media:Biotium_GelRedProductProtocol.pdf]]
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| ==26 October 2010 - Gel Test==
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| ===Cast Gel===
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| * small caster: 10cm x 10cm x 0.5cm = 50mL TAE
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| * 50mL TAE + 1g agarose in 250mL Erlenmyer flask
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| * small stirbar, hotplate set at 200, stir set at 150 for about 10 min, until clear
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| * let cool (about 5 min)
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| * add 5µL GelRed 10,000X, swirl
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| * pour into caster, set comb
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| * let set (about 5min)
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| ===Load Gel===
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| * 4µL Tara Ladder (T)
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| * 4µL Justin Ladder (J)
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| * 2µL Sample (H6) (added minute after applying current)
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| * [[Image:20101026 GelSchematic.jpg|400 px]]
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|
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| ===Run Gel=== | | * comments |
| * 110V, 20 min | | :: [[Image:20130110_PCRa.png|800 px]] |
| * 150V, 10 min | | |
| ===Image Gel=== | | |
| * Ladders show! | | ===Gel=== |
| *: both Tara's and Justin's
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| * [[Image:20101026_Full.TIF|300 px]]
| | {| {{table}} style="text-align: center" |
| * Sample is very faint, may be due to smaller volume placed
| | |- |
| * ~200-300bp fragment
| | ! Pour !! Load !! Run |
| * (Lance et al 2009, says Scun2 has amplicon length of 131-191) | | |- |
| * [[Image:20101026_Zoom.TIF|300 px]]
| | | 270 mL 1x TAE + 5.4 g agarose || 2 µL gel load dye + 4 µL PCR product || 160 V |
| * do another gel with subset of PCR product, full volume | | |- |
| | | 27 µL GelRed || 6 µL ladder || 45 minutes |
| | |} |
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| | :: [[Image:20130110_Gela.png|800 px]] |
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| | * mispriming everywhere - primer pair not usable for CNHY |
| | * lots of mispriming, but may be able to use conditions indicated by orange face ([[Image:orangefrownyface.png|20 px]]), if needed, under stringent conditions |
| | * favorable conditions indicated by [[Image:greenhappyface.png|25 px]] |
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| | |
| | ===Fragment Analysis Submission=== |
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| | * UAGC Submission# ???? |
| | :: [[Image:20130227_Frag.png|900 px]] |
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| | |
| | * FedEx Tracking# [http://www.fedex.com/Tracking?language=english&cntry_code=us&tracknumbers=801124151779 801124151779] |
| | :: [[Image:20130227_FedEx.png|700 px]] |
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| | |
| | * FedEx picked up ____pm, 26 February |
| | * FedEx delivered ____am, 27 February |
| | * UAGC received ____am, 27 February |
| | * UAGC completed ____am, 28 February |
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| ==28 October 2010 - Gel PCR Product (Scun2 F-R)==
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| ===Cast Gel===
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| * medium caster: 20cm x 13cm x 0.5cm = 130mL TAE
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| * 130mL TAE + 2.6g (2%) agarose in 250mL Erlenmyer flask
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| * small stirbar, hotplate set at 200, stir set at 150, for about 15min, until clear
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| * let cool (about 5 min)
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| * add 13µL GelRed 10,000X, swirl
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| * pour into caster, set 36-tooth comb
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| * let set (about 10min)
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| ===Load Gel===
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| * 4µL Ladder
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| * 4µL Sample w/ loading dye
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| * [[Image:20101028 GelSchematic.jpg|800 px]]
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|
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| ===Run Gel===
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| * 110V, 20 min
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| * 150V, 10 min
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| ===Image Gel===
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| * Ladders show!
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Tara's Lab Notebook
