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

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__TOC__
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==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
 
# F - R
 
# F - R pigtail
* comments
# F M13 - R
:: [[Image:20130110_PCRa.png|800 px]]
# F M13 - R pigtail
 
* If they don't hinder efforts or change optimization decisions, then may make optimization of multiplexes cheaper!
 
==11 October 2010 - PCR Thermalcycler Testing==
===Gel===
===Purpose===
 
* 10μL in each well of full-skirt PCR plate, PCR seal
{| {{table}} style="text-align: center"
* First set-up of thermalcycler, established User, Protocols, Plates and Masters
|-
* Data: 20101011_TKL_CyclerTest.tad
! Pour !! Load !! Run
===Results===
|-
* Evaporation all around edges
| 270 mL 1x TAE + 5.4 g agarose || 2 µL gel load dye + 4 µL PCR product || 160 V
* Try using silicone mat?
|-
===Purpose===
| 27 µL GelRed || 6 µL ladder || 45 minutes
* 10μL in each well of full-skirt PCR plate, PCR seal, 1 silicone mat
|}
* Data: 20101011_TKL_CyclerTest1Mat.tad
 
===Results===
 
* Still evaporation around edges, but better
:: [[Image:20130110_Gela.png|800 px]]
* Try using 2 silicone mats?
 
==12 October 2010 - PCR Thermalcycler Testing==
 
===Purpose===
* mispriming everywhere - primer pair not usable for CNHY
* 10μL in each well of full-skirt PCR plate, PCR seal, 2 silicone mats
* lots of mispriming, but may be able to use conditions indicated by orange face ([[Image:orangefrownyface.png|20 px]]), if needed, under stringent conditions
* Data: 20101012_TKL_CyclerTest2Mat.tad
* favorable conditions indicated by [[Image:greenhappyface.png|25 px]]
===Results===
 
* No evaporation!
 
* Silicone mats got stuck on lid
===Fragment Analysis Submission===
* Try using 2 silicone mats with tape?
 
* Try getting samples of different brand of silicone mats, different shape, no holes?
 
==18 October 2010 - PCR Thermalcycler Testing==
* UAGC Submission# ????
===Purpose===
:: [[Image:20130227_Frag.png|900 px]]
* 10µL in each well of full-skirt PCR plate, PCR seal, 2 silicone mats, lab tape on opposite sides
 
* Data: 20102018_TKL_CyclerTest2MatTape.tad
 
===Results===
* FedEx Tracking# [http://www.fedex.com/Tracking?language=english&cntry_code=us&tracknumbers=801124151779 801124151779]
* H6, H7 evaporated, but I think it's workable
:: [[Image:20130227_FedEx.png|700 px]]
* Plan to do first optimization PCR tomorrow!
 
==18 October 2010 - Primer Rehydration, Scun2
 
===Purpose===
* FedEx picked up ____pm, 26 February
* Rehydrate Scun2 primers for testing tomorrow
* FedEx delivered ____am, 27 February
# Scun2-F
* UAGC received ____am, 27 February
# Scun2-R
* UAGC completed ____am, 28 February
# Scun2-F-M13F(-21)
 
# Scun2-R-pigtail
 
===Calculations===
 
*


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Revision as of 09:12, 23 May 2013

Tara's Lab Notebook <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page


CNTI Acos5

PCR

  • comments
File: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


File: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# ????
File:20130227 Frag.png


File: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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