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

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==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?
 
-
==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
 
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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!
 
-
==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
 
-
==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!
 
-
==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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==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==
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===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|400 px]] [[Image:20101025_Bottom.TIF|400 px]]
+
-
===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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==26 October 2010 - GelRed info==
+
===PCR===
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* Yesterday, used 27µL 3x Gel Red into warm agarose/TAE mix (precast)
+
* [[Image:20120110_PCRa.png|800 px]]
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* Biotium's Product Information sheet on GelRed: [[Media:Biotium_GelRedProductProtocol.pdf‎]]
+
 
 +
 
 +
===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:20120110_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===
 +
 
 +
 
 +
:: [[Image:20130226_Frag.png|900 px]]
 +
 
 +
 
 +
* FedEx Tracking# [http://www.fedex.com/Tracking?language=english&cntry_code=us&tracknumbers=801124151778 801124151778]
 +
:: [[Image:20130226_FedEx.png|700 px]]
 +
 
 +
 
 +
* UAGC Submission# PPTX
 +
 
 +
 
 +
* FedEx picked up 2:44pm, 26 February
 +
* FedEx delivered 9:29am, 27 February
 +
* UAGC received 10:58pm, 27 February
 +
* UAGC completed 10:41am, 28 February
-
==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]]
 
-
===Run Gel===
 
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* 110V, 20 min
 
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* 150V, 10 min
 
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===Image Gel===
 
-
*
 

Revision as of 19:02, 12 March 2013

Tara's Lab Notebook Main project page

Contents


CNTI Acos5

PCR


Gel

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


  • mispriming everywhere - primer pair not usable for CNHY
  • lots of mispriming, but may be able to use conditions indicated by orange face (), if needed, under stringent conditions
  • favorable conditions indicated by


Fragment Analysis Submission



  • UAGC Submission# PPTX


  • FedEx picked up 2:44pm, 26 February
  • FedEx delivered 9:29am, 27 February
  • UAGC received 10:58pm, 27 February
  • UAGC completed 10:41am, 28 February




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