Dinh/NOTES/2009-12-4: Difference between revisions
Jump to navigation
Jump to search
>Dinh |
>Dinh mNo edit summary |
||
Line 3: | Line 3: | ||
==Materials== | ==Materials== | ||
*Quan-iT ssDNA Assay Kit (cat. # Q32851, containing: | |||
*Quant-iT ssDNA reagent, 200x solution in DMSO | *Quant-iT ssDNA reagent, 200x solution in DMSO | ||
*Quant-iT buffer | *Quant-iT buffer | ||
Line 10: | Line 10: | ||
*Qubit fluorometer Invitrogen Cat. Q32857 | *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 | *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== | ==Starting Notes== | ||
Line 55: | Line 55: | ||
==Protocol== | ==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 | 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== | ==Results== | ||
{| border="1" | {| border="1" | ||
!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 | |||
|- | |||
|} | |} | ||
Revision as of 01:39, 5 December 2009
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 |