Matt:LabNotes/2014-3-5
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Agi26k0gap Padlock Probe Prep
- Taking column purified 0gap probes from Matt:LabNotes/2013-5-16
- Run 3 gels each with 60ul
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 |
- 20X diluated sample concentration is __ng/ul
- Starting sample concentration: __ng/ul -> total yield about __ug/(193nt*325 Da/nt) = ___ pmole