Matt:LabNotes/2014-6-27: Difference between revisions

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>Mzcai
(Created page with "==Agi26k0gap Probe Production Continued== *Continued from: Matt:LabNotes/2014-6-24 ===PAGE Size Selection=== *Run 4 gels of ~3.2ug ssDNA in each **Corresponds to 22.5ul ...")
 
>Mzcai
 
(3 intermediate revisions by the same user not shown)
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*Ran at 220V for 30min
*Ran at 220V for 30min
*Cut out brightest band below 200bp
*Cut out brightest band below 200bp
[[File:2014-06-27_Agi26k0gapSizeSelectGel1.jpg]]<br>


[[File:2014-06-27_Agi26k0gapSizeSelectGel2.jpg]]<br>
[[File:2014-06-27_Agi26k0gapSizeSelectGel3.jpg]]<br>
[[File:2014-06-27_Agi26k0gapSizeSelectGel4.jpg]]
===EtOH Precipitation===
===EtOH Precipitation===
*Put cut out gel in 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT.
*Put cut out gel in 0.5mL tube (with holes 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.
*Transfered the gel remaining in 0.5 mL tube to 2.0 mL tube below with pipette tip.
*Added 450 ul of 1X TE buffer
*Added 900 ul of 1X TE buffer
*Vortexed for 45-60 min at 37 C in incubator
*Vortexed for 60 min at 37 C in incubator
*Centrifuged at 15,000 rpm for 3 min at RT
*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 clear spnt. to 8 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 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)
*Transfered spnt to fresh 1.5 mL tube (~450ul per tube)
*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, 45ul of 3M NaoAc pH 5.2
*Vortexed and placed the 4 tubes at -80C
*Vortexed and placed the 8 tubes at -80C
*Spun 8 tubes at 10,000rpm at 4C for 30min
*Discard supernatant and add 650ul 75% EtOH and spin at 14,000rpm at 4C for 5min
*Discard supernatant and let dry in hood for 10min
*Resuspend each tube with 10ul (80ul total)
 
===TBU Gel Quantification===
*Diluted Agi26k0gap 10X
**2ul Agi26k0gap + 8ul H2O + 10ul 2X TBE-Urea buffer
*Made DNA Low Mass Ladder with 0.5, 1, and 2 ul
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''1'''
| align="center" style="background:#f0f0f0;"|'''2'''
| align="center" style="background:#f0f0f0;"|'''3'''
| align="center" style="background:#f0f0f0;"|'''4'''
| align="center" style="background:#f0f0f0;"|'''5'''
| align="center" style="background:#f0f0f0;"|'''6'''
| align="center" style="background:#f0f0f0;"|'''7'''
|-
| Sample||0.5ul DNA Ladder||1ul DNA Ladder||2ul Sample||4ul Sample||6ul Sample||8ul Sample||2ul DNA Ladder
|}
[[File:2014-06-30_Agi26k0gapTBUGelQuant.jpg]]
 
*[[Media:2014-06-30_Agi26k0gapTBUGelQuantAnalysis.xlsx | Quantification Analysis]]
 
*Agi26k0gap Concentration: 56.9 ng/ul
*Total Mass: 56.9 ng/ul * 80ul = 4.55 ug
*Total Amount: 4.55ug /(193nt*325 Da/nt) = 72.6 pmole (908nM)

Latest revision as of 23:38, 1 July 2014

Agi26k0gap Probe Production Continued[edit]

PAGE Size Selection[edit]

  • Run 4 gels of ~3.2ug ssDNA in each
    • Corresponds to 22.5ul
Components 1X Volume 4X Volume
Enzyme digested probes 22.5 90
TBE-Urea Buffer 2X 50 200
Water 27.5 110
Total 100 400
Components 4X Volume
Low Mass Ladder 4
TBE-Urea Buffer 2X 20
H2O 16
Total 40
  • Ran at 220V for 30min
  • Cut out brightest band below 200bp

File:2014-06-27 Agi26k0gapSizeSelectGel1.jpg

File:2014-06-27 Agi26k0gapSizeSelectGel2.jpg

File:2014-06-27 Agi26k0gapSizeSelectGel3.jpg

File:2014-06-27 Agi26k0gapSizeSelectGel4.jpg

EtOH Precipitation[edit]

  • Put cut out gel in 0.5mL tube (with holes 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 60 min at 37 C in incubator
  • Centrifuged at 15,000 rpm for 3 min at RT
  • Transfered the clear spnt. to 8 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 (~450ul per tube)
  • Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 8 tubes at -80C
  • Spun 8 tubes at 10,000rpm at 4C for 30min
  • Discard supernatant and add 650ul 75% EtOH and spin at 14,000rpm at 4C for 5min
  • Discard supernatant and let dry in hood for 10min
  • Resuspend each tube with 10ul (80ul total)

TBU Gel Quantification[edit]

  • Diluted Agi26k0gap 10X
    • 2ul Agi26k0gap + 8ul H2O + 10ul 2X TBE-Urea buffer
  • Made DNA Low Mass Ladder with 0.5, 1, and 2 ul
Lane 1 2 3 4 5 6 7
Sample 0.5ul DNA Ladder 1ul DNA Ladder 2ul Sample 4ul Sample 6ul Sample 8ul Sample 2ul DNA Ladder

File:2014-06-30 Agi26k0gapTBUGelQuant.jpg

  • Agi26k0gap Concentration: 56.9 ng/ul
  • Total Mass: 56.9 ng/ul * 80ul = 4.55 ug
  • Total Amount: 4.55ug /(193nt*325 Da/nt) = 72.6 pmole (908nM)