Matt:LabNotes/2014-6-27: Difference between revisions
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>Mzcai |
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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. | ||
Line 42: | Line 48: | ||
*Vortexed for 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 | *Transfered spnt to fresh 1.5 mL tube (~450ul per tube) | ||
*Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, | *Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2 | ||
*Vortexed and placed the | *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]
- Continued from: Matt:LabNotes/2014-6-24
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)