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

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Image Gel)
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==
+
==Task==
-
* Ordered first set of primers
+
* here
-
* Scun2 and Scun22 (highly variable simple motifs)
+
* here
-
** Lance et al (2009) Conservation Genetics Resources, Sceloporus undulatus
+
** in here
-
* Forward and reverse as given in publication, also reverse with pigtail (GTTTCTT) at 5' end, also forward with M13F(-21) tag (TGTAAAACGACGGCCAGT)
+
# numbered
-
* [[Image:Luckau_Primer20100927.jpg|500 px]]
+
# again
-
* Try all four combinations
+
-
# F - R
+
-
# F - R pigtail
+
-
# F M13 - R
+
-
# 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==
+
-
===Purpose===
+
-
* 10μL in each well of full-skirt PCR plate, PCR seal
+
-
* First set-up of thermalcycler, established User, Protocols, Plates and Masters
+
-
* Data: 20101011_TKL_CyclerTest.tad
+
-
===Results===
+
-
* Evaporation all around edges
+
-
* Try using silicone mat?
+
-
===Purpose===
+
-
* 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
+
-
* Try using 2 silicone mats?
+
-
==12 October 2010 - PCR Thermalcycler Testing==
+
-
===Purpose===
+
-
* 10μL in each well of full-skirt PCR plate, PCR seal, 2 silicone mats
+
-
* Data: 20101012_TKL_CyclerTest2Mat.tad
+
-
===Results===
+
-
* No evaporation!
+
-
* 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?
+
-
==18 October 2010 - PCR Thermalcycler Testing==
+
-
===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
+
-
===Results===
+
-
* H6, H7 evaporated, but I think it's workable
+
-
* Plan to do first optimization PCR tomorrow!
+
-
==18 October 2010 - Primer Rehydration, Scun2==
+
-
===Purpose===
+
-
* Rehydrate Scun2 primers for testing tomorrow
+
-
# Scun2-F
+
-
# Scun2-R
+
-
# 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 - Ladder Mix==
+
===Subsection===
-
===Manufacturer specifications===
+
* here
-
* 0.1µg ladder / mm width of well
+
-
* STK at 1.0µg/µL
+
-
* [[Media:Invitrogen_1KbLadderProductManual.pdf]]
+
-
===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
+
-
==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)
 
-
===Load Gel===
 
-
*[[Image:BlankGelSchematicTop.jpg|600 px]]
 
-
===Run Gel===
 
-
* use top volt-box
 
-
* Power On
 
-
*: DC On
 
-
*: Voltmeter: push to right
 
-
*: Adjust red knob to 90-120V (using red scale of left-side gauge)
 
-
* 90V 3p-4:30p
 
-
*: 120V 4:30p-4:45p
 
-
===Image Gel===
 
-
* nothing...
 
-
*: [[Image:20101025_Top.TIF|300 px]] [[Image:20101025_Bottom.TIF|300 px]]
 
-
 
-
===What Happened?===
 
-
* UV light?
 
-
** should be working, when turned off, image black, when turned on, can see gel (but only agarose gel)
 
-
* Gel Stain?
 
-
** used as according to Clark's protocol (10% volume of 3x Gel Red)
 
-
** check product protocol
 
-
* Next:
 
-
** try a bunch of different trials of ladder, gel stain, on small gel
 
-
 
-
==26 October 2010 - GelRed info==
 
-
* Yesterday, used 27µL 3x Gel Red into warm agarose/TAE mix (precast)
 
-
* Biotium's Product Information sheet on GelRed: [[Media:Biotium_GelRedProductProtocol.pdf‎]]
 
-
 
-
==26 October 2010 - Gel Test==
 
-
===Cast Gel===
 
-
* small caster: 10cm x 10cm x 0.5cm = 50mL TAE
 
-
* 50mL TAE + 1g agarose in 250mL Erlenmyer flask
 
-
* small stirbar, hotplate set at 200, stir set at 150 for about 10 min, until clear
 
-
* let cool (about 5 min)
 
-
* add 5µL GelRed 10,000X, swirl
 
-
* pour into caster, set comb
 
-
* let set (about 5min)
 
-
===Load Gel===
 
-
* 4µL Tara Ladder (T)
 
-
* 4µL Justin Ladder (J)
 
-
* 2µL Sample (H6) (added minute after applying current)
 
-
* [[Image:20101026 GelSchematic.jpg|400 px]]
 
-
 
-
===Run Gel===
 
-
* 110V, 20 min
 
-
* 150V, 10 min
 
-
===Image Gel===
 
-
* Ladders show!
 
-
*: both Tara's and Justin's
 
-
* [[Image:20101026_Full.TIF|300 px]]
 
-
* Sample is very faint, may be due to smaller volume placed
 
-
* ~200-300bp fragment
 
-
* (Lance et al 2009, says Scun2 has amplicon length of 131-191)
 
-
* [[Image:20101026_Zoom.TIF|300 px]]
 
-
* do another gel with subset of PCR product, full volume
 
-
 
-
==28 October 2010 - Gel Scun2 F-R==
 
-
===Cast Gel===
 
-
* medium caster: 20cm x 13cm x 0.5cm = 130mL TAE
 
-
* 130mL TAE + 2.6g (2%) agarose in 250mL Erlenmyer flask
 
-
* small stirbar, hotplate set at 200, stir set at 150, for about 15min, until clear
 
-
* let cool (about 5 min)
 
-
* add 13µL GelRed 10,000X, swirl
 
-
* pour into caster, set 36-tooth comb
 
-
* let set (about 10min)
 
-
===Load Gel===
 
-
* 4µL Ladder
 
-
* 4µL Sample w/ loading dye
 
-
* [[Image:20101028 GelSchematic.jpg|800 px]]
 
-
 
-
===Run Gel===
 
-
* 110V, 2 hrs
 
-
 
-
===Image Gel===
 
-
* Nada, zip, zilch ... poopy
 
-
* [[Image:20101028_Full.TIF|350 px]]
 
-
* [[Image:20101028_Left.TIF|350 px]] [[Image:20101028_Right.TIF|350 px]]
 
-
* WTF?
 

Revision as of 19:05, 28 October 2010

Tara's Lab Notebook Main project page

Contents

Task

  • here
  • here
    • in here
  1. numbered
  2. again

Subsection

  • here



Personal tools