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| |style="background-color: #BCED91"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Tara's Lab Notebook</span> | | |style="background-color: #BCED91"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Tara's Lab Notebook</span> |
| |style="background-color: #9DB68C" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | | |style="background-color: #9DB68C" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} |
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| <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> |
| __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!
| |
| ==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?
| |
| ==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
| |
| ===Results===
| |
| * H6, H7 evaporated, but I think it's workable
| |
| * Plan to do first optimization PCR tomorrow!
| |
| ==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===
| |
| * 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
| |
| ==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
| |
| ===Wet Work===
| |
| * 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
| |
| ==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!
| |
| ===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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|
| |
|
| ==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
| |
| * [[Media:Invitrogen_1KbLadderProductManual.pdf]]
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| ===Other Knowns===
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| * well width (24-tooth comb) = 6mm
| |
| * Jeremy (Bohonak lab) uses 5µL PCR product + 1.5µL dye
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| ===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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|
| |
|
| ==25 October 2010 - Gel Scun2 F-R== | | ==CNTI Acos5== |
| ===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
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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)
| |
| ===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
| |
| ===Image Gel===
| |
| * nothing...
| |
| *: [[Image:20101025_Top.TIF|400 px]] [[Image:20101025_Bottom.TIF|400 px]]
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|
| |
|
| ===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== | | ===PCR=== |
| * Yesterday, used 27µL 3x Gel Red into warm agarose/TAE mix (precast) | | |
| * Biotium's Product Information sheet on GelRed: [[Media:Biotium_GelRedProductProtocol.pdf]] | | |
| | * comments |
| | :: [[Image:20130110_PCRa.png|800 px]] |
| | |
| | |
| | ===Gel=== |
| | |
| | {| {{table}} style="text-align: center" |
| | |- |
| | ! Pour !! Load !! Run |
| | |- |
| | | 270 mL 1x TAE + 5.4 g agarose || 2 µL gel load dye + 4 µL PCR product || 160 V |
| | |- |
| | | 27 µL GelRed || 6 µL ladder || 45 minutes |
| | |} |
| | |
| | |
| | :: [[Image:20130110_Gela.png|800 px]] |
| | |
| | |
| | * 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]] |
| | |
| | |
| | ===Fragment Analysis Submission=== |
| | |
| | |
| | * UAGC Submission# ???? |
| | :: [[Image:20130227_Frag.png|900 px]] |
| | |
| | |
| | * FedEx Tracking# [http://www.fedex.com/Tracking?language=english&cntry_code=us&tracknumbers=801124151779 801124151779] |
| | :: [[Image:20130227_FedEx.png|700 px]] |
| | |
| | |
| | * FedEx picked up ____pm, 26 February |
| | * FedEx delivered ____am, 27 February |
| | * UAGC received ____am, 27 February |
| | * UAGC completed ____am, 28 February |
|
| |
|
| ==26 October 2010 - Gel Test==
| |
| ===Cast Gel===
| |
| * small caster: 10cm x 10cm x 0.5cm = 50mL TAE
| |
| * 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
| |
| * let cool (about 5 min)
| |
| * add 5µL GelRed 10,000X, swirl
| |
| * pour into caster, set comb
| |
| * let set (about 5min)
| |
| ===Load Gel===
| |
| * 4µL Tara Ladder (T)
| |
| * 4µL Justin Ladder (J)
| |
| * 2µL Sample (H6) (added minute after applying current)
| |
| * [[Image:20101026 GelSchematic.jpg|400 px]]
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|
| |
|
| ===Run Gel===
| |
| * 110V, 20 min
| |
| * 150V, 10 min
| |
| ===Image Gel===
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| *
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|
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|