User:Tara K. Luckau/Notebook/Team ConGen/2010/10/18: Difference between revisions

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* H6, H7 evaporated, but I think it's workable
* H6, H7 evaporated, but I think it's workable
* Plan to do first optimization PCR tomorrow!
* Plan to do first optimization PCR tomorrow!
==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
==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




Revision as of 17:17, 28 October 2010

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PCR Thermalcycler Testing

2 Mats w/ Tape

Description

  • 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!

Primer Rehydration, Scun2

Purpose

  • Rehydrate Scun2 primers for testing tomorrow
  1. Scun2-F
  2. Scun2-R
  3. Scun2-F-M13F(-21)
  4. 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

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
  • (C1) (V1) = (C2) (V2)
    (2.5mM) (5000µL) = (100mM) (V2)
    V2 = 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




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