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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==CNTI Acos5==
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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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===PCR===
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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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* comments
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# F M13 - R
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:: [[Image:20130110_PCRa.png|800 px]]
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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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===Gel===
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===Purpose===
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* 10μL in each well of full-skirt PCR plate, PCR seal
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{| {{table}} style="text-align: center"
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* First set-up of thermalcycler, established User, Protocols, Plates and Masters
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|-
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* Data: 20101011_TKL_CyclerTest.tad
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! Pour !! Load !! Run
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===Results===
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|-
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* Evaporation all around edges
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| 270 mL 1x TAE + 5.4 g agarose || 2 µL gel load dye + 4 µL PCR product || 160 V
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* Try using silicone mat?
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|-
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===Purpose===
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| 27 µL GelRed || 6 µL ladder || 45 minutes
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* 10μL in each well of full-skirt PCR plate, PCR seal, 1 silicone mat
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|}
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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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:: [[Image:20130110_Gela.png|800 px]]
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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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* mispriming everywhere - primer pair not usable for CNHY
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* 10μL in each well of full-skirt PCR plate, PCR seal, 2 silicone mats
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* lots of mispriming, but may be able to use conditions indicated by orange face ([[Image:orangefrownyface.png|20 px]]), if needed, under stringent conditions
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* Data: 20101012_TKL_CyclerTest2Mat.tad
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* favorable conditions indicated by [[Image:greenhappyface.png|25 px]]
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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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===Fragment Analysis Submission===
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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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* UAGC Submission# ????
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===Purpose===
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:: [[Image:20130227_Frag.png|900 px]]
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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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* FedEx Tracking# [http://www.fedex.com/Tracking?language=english&cntry_code=us&tracknumbers=801124151779 801124151779]
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* H6, H7 evaporated, but I think it's workable
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:: [[Image:20130227_FedEx.png|700 px]]
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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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* FedEx picked up ____pm, 26 February
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* Rehydrate Scun2 primers for testing tomorrow
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* FedEx delivered ____am, 27 February
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# Scun2-F
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* UAGC received ____am, 27 February
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# Scun2-R
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* UAGC completed ____am, 28 February
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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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* (C1)(V1)=(C2)(V2)
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*: (2.5mM)(5000µL)=(100mM)(V2)
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*: V2=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]]
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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 H2O
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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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Current revision

Tara's Lab Notebook Main project page

Contents


CNTI Acos5

PCR

  • comments
Image:20130110 PCRa.png


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


Image:20130110 Gela.png


  • 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# ????
Image:20130227 Frag.png


Image:20130227 FedEx.png


  • 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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