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
Quant-iT ssDNA | ' | ' | ' | ' | Orig | Orig | ' |
Qubit Reading(ng/mL) | uL used | Dilution | Sample (ng/mL) | Sample Dilution | Sample (ng/uL) | Average (ng/ul) | |
Standard | 104 | 2 | 100 | 10400 | |||
1a | 209 | 4 | 50 | 10450 | 10 | 104.5 | |
1b | 20.3 | 4 | 50 | 1015 | 100 | 101.5 | 103 |
2a | 111 | 4 | 50 | 5550 | 10 | 55.5 | |
2b | 11 | 4 | 50 | 550 | 100 | 55 | 55.25 |
3a | 48.4 | 4 | 50 | 2420 | 33.33 | 80.67 | |
3b | 5.17 | 4 | 50 | 258.5 | 333.33 | 86.17 | 83.42 |
4a | 108 | 4 | 50 | 5400 | 10 | 54 | |
4b | 9.56 | 4 | 50 | 478 | 100 | 47.8 | 50.9 |
5a | 3.3 | 2 | 100 | 330 | 1 | 0.33 | |
5a | 3.2 | 2 | 100 | 320 | 1 | 0.32 | 0.33 |
6a | 47.9 | 4 | 50 | 2395 | 10 | 23.95 | |
6b | 4.6 | 4 | 50 | 230 | 100 | 23 | 23.48 |