Matt:LabNotes/2014-8-7
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Agi26k0gap Probe Production Continued[edit]
- Continued from: Matt:LabNotes/2014-8-4
PAGE Size Selection[edit]
- Run 6 gels of ~2.8ug ssDNA in each
- Corresponds to 20.7ul in each gel
Components | 1X Volume | 6X Volume |
Enzyme digested probes | 20.7 | 124 |
TBE-Urea Buffer 2X | 50 | 300 |
Water | 29.3 | 176 |
Total | 100 | 600 |
Components | 6X Volume |
Low Mass Ladder | 6 |
TBE-Urea Buffer 2X | 30 |
H2O | 24 |
Total | 60 |
- Ran at 220V for 30min
- Cut out brightest band below 200bp
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 12 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 12 tubes at -80C for 3 hrs
- Spun 12 tubes at 10,000rpm at 4C for 30min
- Discard supernatant and add 650ul 80% EtOH and spin at 14,000rpm at 4C for 5min
- Discard supernatant and let dry in hood for 10min
- Resuspend each tube with 10ul (120ul 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:2014-08-08 Agi26k0gapGelQuant.jpg
- Agi26k0gap Concentration: 38.32 ng/ul
- Total Mass: 38.32 ng/ul * 120ul = 4.60 ug
- Total Amount: 4.60ug /(193nt*325 Da/nt) = 73.3 pmole (611nM)