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

From OpenWetWare
Jump to: navigation, search
(25 October 2010 - Ladder Mix)
(38 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
+
 
# 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===
 
* Scun2-F
 
** 28.3nmol + 141.5 Low TE = 200µM STK
 
* Scun2-R
 
** 29.2nmol + 146µL Low TE = 200µM STK
 
* Scun2-F-M13F(-21)
 
** 26.6nmol + 133µL Low TE = 200µM STK
 
* Scun2-R-pigtail
 
** 30.5nmol + 152.5µL Low TE = 200µM STK
 
* stored overnight in fridge to rehydrate completely before making dilution to 20µM
 
==18 October 2010 - dNTP Mix==
 
===Purpose===
 
* Mix dNTPs and aliquot for PCR
 
===Calculations===
 
* Have: Invitrogen 100mM dNTP Set (catalog# 10297-018) (250µL of 25µmol in H2O @ 100mM each)
 
* Want: 10mM dNTP mix (2.5mM each), 5 aliquots of 1000µL
 
* (C<sub>1</sub>) (V<sub>1</sub>) = (C<sub>2</sub>) (V<sub>2</sub>)
 
*: (2.5mM) (5000µL) = (100mM) (V<sub>2</sub>)
 
*: 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)
 
* Used NanoPure H20 from room 325
 
* 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==
 
* [[Image:Luckau_PCR20101019.jpg|700 px]]
 
* DNA = SCOC CPN 82
 
* 2 mats with lab tape, no evaporation!
 
==21 October 2010 - Gel Scun2 F-R==
 
===Make 1x TAE===
 
* 20mL 50x TAE + 980 mL NanoPure H<sub>2</sub>O
 
===Cast Gel===
 
* 100mL 1x TAE + 1.5g agarose
 
* large stirbar, hotplate set at 150 for about 30 min
 
* let cool about 20 min
 
* not enough volume! FAIL!
 
===Next Try===
 
* 270mL TAE + 4.05g agarose
 
* (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)
 
==25 October 2010 - Gel Scun2 F-R==
 
===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
 
* let cool in dark for about 30 min
 
* pour into caster, set 2 24-tooth combs, cover with cardboard
 
* let cool in dark (had to do office hours, sat for about 2 hours)
 
==25 October 2010 - Ladder Mix==
 
===Manufacturer specifications===
 
* 0.1µg ladder / mm width of well
 
* STK at 1.0µg/µL
 
===Other Knowns===
 
* well width (24-tooth comb) = 6mm
 
* Jeremy (Bohonak lab) uses 5µL PCR product + 1.5µL dye
 
===Calculations===
 
* 6mm x (0.1µg ladder)/(1mm width) x (1µL/1µg) = 0.6µL ladder per well
 
* Per well: 0.6µL ladder + 4.4µL H<sub>2</sub>O + 1.5µL loading dye = 6.5µL total ladder mix
 
* (loading dye = xylene cyanol  and bromophenol, made by Bohonak lab)
 
===Make Ladder Mix===
 
* 120µL ladder + 880µL H<sub>2</sub>O + 300µL loading dye = 1300µL ladder mix
 
  
  

Revision as of 09:12, 23 May 2013

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


CNTI Acos5

PCR

  • comments
800 px


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


800 px


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


Fragment Analysis Submission

  • UAGC Submission# ????
900 px


700 px


  • FedEx picked up ____pm, 26 February
  • FedEx delivered ____am, 27 February
  • UAGC received ____am, 27 February
  • UAGC completed ____am, 28 February