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

From OpenWetWare
Jump to navigationJump to search
No edit summary
No edit summary
(39 intermediate revisions by the same user not shown)
Line 7: Line 7:
<!-- ##### 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==
 
* 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
* [[Image:20120110_PCRa.png|800 px]]
# F - R
 
# F - R pigtail
 
# F M13 - R
===Gel===
# F M13 - R pigtail
 
* If they don't hinder efforts or change optimization decisions, then may make optimization of multiplexes cheaper!
{| {{table}} style="text-align: center"
==11 October 2010 - PCR Thermalcycler Testing==
|-
===Purpose===
! Pour !! Load !! Run
* 10μL in each well of full-skirt PCR plate, PCR seal
|-
* First set-up of thermalcycler, established User, Protocols, Plates and Masters
| 270 mL 1x TAE + 5.4 g agarose || 2 µL gel load dye + 4 µL PCR product || 160 V
* Data: 20101011_TKL_CyclerTest.tad
|-
===Results===
| 27 µL GelRed || 6 µL ladder || 45 minutes
* Evaporation all around edges
|}
* Try using silicone mat?
 
===Purpose===
 
* 10μL in each well of full-skirt PCR plate, PCR seal, 1 silicone mat
[[Image:20120110_Gela.png|800 px]]
* Data: 20101011_TKL_CyclerTest1Mat.tad
 
===Results===
* mispriming everywhere - primer pair not usable for CNHY
* Still evaporation around edges, but better
* lots of mispriming, but may be able to use conditions indicated by orange face ([[Image:orangefrownyface.png|20 px]]), if needed, under stringent conditions
* Try using 2 silicone mats?
* favorable conditions indicated by [[Image:greenhappyface.png|25 px]]
==12 October 2010 - PCR Thermalcycler Testing==
 
===Purpose===
 
* 10μL in each well of full-skirt PCR plate, PCR seal, 2 silicone mats
===Fragment Analysis Submission===
* Data: 20101012_TKL_CyclerTest2Mat.tad
 
===Results===
 
* No evaporation!
:: [[Image:20130226_Frag.png|900 px]]
* Silicone mats got stuck on lid
 
* Try using 2 silicone mats with tape?
 
* Try getting samples of different brand of silicone mats, different shape, no holes?
* FedEx Tracking# [http://www.fedex.com/Tracking?language=english&cntry_code=us&tracknumbers=801124151778 801124151778]
==18 October 2010 - PCR Thermalcycler Testing==
:: [[Image:20130226_FedEx.png|700 px]]
===Purpose===
 
* 10µL in each well of full-skirt PCR plate, PCR seal, 2 silicone mats, lab tape on opposite sides
 
* Data: 20102018_TKL_CyclerTest2MatTape.tad
* UAGC Submission# PPTX
===Results===
 
* H6, H7 evaporated, but I think it's workable
 
* Plan to do first optimization PCR tomorrow!
* FedEx picked up 2:44pm, 26 February
==18 October 2010 - Primer Rehydration, Scun2
* FedEx delivered 9:29am, 27 February
===Purpose===
* UAGC received 10:58pm, 27 February
* Rehydrate Scun2 primers for testing tomorrow
* UAGC completed 10:41am, 28 February
# Scun2-F
 
# Scun2-R
 
# Scun2-F-M13F(-21)
 
# Scun2-R-pigtail
===Calculations===
*


<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Revision as of 16:02, 12 March 2013

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


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




Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Tara_K._Luckau/Notebook/Team_ConGen/Entry_Base&oldid=682709"