Dinh/NOTES/2009-12-4
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Quant-iT(TM) ssDNA Assay Kit
Materials
- Quan-iT ssDNA Assay Kit (cat. # Q32851, containing:
*Quant-iT ssDNA reagent, 200x solution in DMSO *Quant-iT buffer *Oligonucleotide standard #1 (0ng/ul in TE Buffer) *Oligonucleotide standard #2 (20ng/ul in TE Buffer)
- Qubit fluorometer Invitrogen Cat. Q32857
- Qubit Assay Tubes (500, Invitrogen Cat. no.Q32856) or Axygen PCR-05-C tubes (VWR, part number 10011-830)*Qubit fluorometer Invitrogen Cat. Q32857
- Sterile, distilled, DNase free water
Starting Notes
- Make sure that all solutions are at room temperature
- Do not hold the assay tubes in your hand before reading
- Do not leave tubes in the fluorometer for longer than necessary as it may warm the assay solution significantly
- Do not leave assay solutions exposed to light
- Do not overexpose the assay solutions to more than 9 readings as solution is not photostable.
- Assay solutions are not stable. Discard after 30min.
Table 1. Solution
Standard/Sample Input | Volume (ul) of standard/sample | Volume (ul) of diluted Working Solution | Final Oligomer Conc. |
---|---|---|---|
Standard #1 | 10 | 190 | 0ng/ul |
Standard #2 | 10 | 190 | 1ng/ul |
Sample #1-6 | 1 | 199 | ??? |
Total working solution required: 2780ul
Sample Information:
- after 1st EtOH PPT, 1020ul
- after Lamda Exonuclease, 1083ul
- after QiaQuick, 420ul, diluted 10/3x
- after USER & DpnII, 518ul
- after size selection & Nanosep, 4047ul
- after EtOH PPT, 20ul, diluted to 0.1x
Protocol
- Set up 2 tubes for standard and 12 tubes for samples (2x each 1-6)
- Label tube lids
- Calculate amount of working solution required.
- Load in appropriate volumes for each labeled tubes
- Vortex for 5 s and spin down all tubes
- Incubate for 2 minutes at room temperature
- Turn on Qubit Fluorometer
- Press HOME and select Quant-iT ssDNA. Press GO to initiate assay
- On calibration screen, select Run New Calibration. Press GO
Or select use last calibration
- Insert standard#1, close lid, press GO
- Remove standard#1, insert standard#2, close lid, press GO
- Remove standard#2.
- Repeat similarly for sample assay tubes.
- Record readings given by the fluorometer.
Results
Sample | 1a | 1b | 2a | 2b | 3a | 3b | 4a | 4b | 5a | 5b | 6a | 6b |
---|---|---|---|---|---|---|---|---|---|---|---|---|
Conc. (ng/ul) | 1a | 1b | 2a | 2b | 3a | 3b | 4a | 4b | 5a | 5b | 6a | 6b |