Matt:LabNotes/2014-3-5: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
(Created page with "==Agi26k0gap Padlock Probe Prep== *Taking column purified 0gap probes from Matt:LabNotes/2013-5-16 *Run 3 gels each with 60ul ===Procedure=== Sample <br> {| {{table}} | ...")
 
>Mzcai
mNo edit summary
Line 61: Line 61:
*Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
*Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
*Vortexed and placed the 6 tubes at -80C overnight
*Vortexed and placed the 6 tubes at -80C overnight
====Day 2====
'''Ethanol Precipitation after PAGE size selection'''
*Spun 6 tubes in -80C at 10,000rpm at 4C for 30min
*Discard supernatant and add 650ul 75% EtOH to wash and spin at 14,000rpm at 4C for 5 min
*Discard supernatant and let dry in hood 10 min
*Resuspended each of the 6 tubes with 10ul H2O (60ul total)
'''TBU Gel Quantification Plan'''
*Dilute 2ul into 20ul
*Mix 1:1 with 2X buffer (20X dilution)
*Load sample wells with 2, 4, 6, 8 ul
*Load Low Mass DNA ladders in 0.5, 1, and 2 ul
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''0.5 ladder'''
| align="center" style="background:#f0f0f0;"|'''1 ladder'''
| align="center" style="background:#f0f0f0;"|''' 2 ul '''
| align="center" style="background:#f0f0f0;"|''' 4 ul '''
| align="center" style="background:#f0f0f0;"|''' 6 ul '''
| align="center" style="background:#f0f0f0;"|''' 8 ul '''
| align="center" style="background:#f0f0f0;"|'''2 ladder'''
|-
| DNA volume||0.5||1||0.1||0.2||0.3||0.4||2
|-
| H2O||1.5||1||0.9||1.8||2.7||3.6||0
|-
| 2X Buffer||2||2||1||2||3||4||2
|-
| Total||4||4||2||4||6||8||4
|-
|
|}
[[650px]]
*20X diluated sample concentration is __ng/ul
*Starting sample concentration: __ng/ul -> total yield about __ug/(193nt*325 Da/nt) = ___ pmole

Revision as of 02:39, 7 March 2014

Agi26k0gap Padlock Probe Prep

Procedure

Sample

Components 1X Volume 3X Volume
Enzyme digested probes (~10ug/180ul) 58 174
TBE-Urea Buffer 2X 58 174
Water 0 0
Total 116 348

3 Low Mass DNA Ladders

Components 3X Volume
Low Mass DNA ladder (1 ug/ul) 3
TBE-Urea Buffer 2X 15
H2O 12
Total 30
  • Ran the 3 gels at 220V for 30min
  • Shake in SYBR Gold for 6+ minutes
  • Cut out band below 200bp

Gel 1:
File:2014-03-05 Agi26k0gapPrep Gel1.jpg

Gel 2:
File:2014-03-05 Agi26k0gapPrep Gel2.jpg

Gel 3:
File:2014-03-05 Agi26k0gapPrep Gel3.jpg

  • Put cut out gel in 0.5mL tube (with hole at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT.
  • Transfered the gel remaining in 0.5 mL tube to 2.0 mL tube below with pipette tip.
  • Added 900 ul of 1X TE buffer
  • Vortexed for 3.5hr at 37 C in incubator
  • Centrifuged at 15,000 rpm for 3 min at RT
  • Transfered the clear spnt. to Nanosep column and centrifuged at 15,000 rpm for 3 min
  • Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
  • Transfered spnt to fresh 1.5 mL tube (volume ~ 400ul/ tube)
  • Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 6 tubes at -80C overnight

Day 2

Ethanol Precipitation after PAGE size selection

  • Spun 6 tubes in -80C at 10,000rpm at 4C for 30min
  • Discard supernatant and add 650ul 75% EtOH to wash and spin at 14,000rpm at 4C for 5 min
  • Discard supernatant and let dry in hood 10 min
  • Resuspended each of the 6 tubes with 10ul H2O (60ul total)

TBU Gel Quantification Plan

  • Dilute 2ul into 20ul
  • Mix 1:1 with 2X buffer (20X dilution)
  • Load sample wells with 2, 4, 6, 8 ul
  • Load Low Mass DNA ladders in 0.5, 1, and 2 ul
' 0.5 ladder 1 ladder 2 ul 4 ul 6 ul 8 ul 2 ladder
DNA volume 0.5 1 0.1 0.2 0.3 0.4 2
H2O 1.5 1 0.9 1.8 2.7 3.6 0
2X Buffer 2 2 1 2 3 4 2
Total 4 4 2 4 6 8 4

650px

  • 20X diluated sample concentration is __ng/ul
  • Starting sample concentration: __ng/ul -> total yield about __ug/(193nt*325 Da/nt) = ___ pmole