Dinh/NOTES/2009-12-4: Difference between revisions
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=Quant-iT(TM) ssDNA Assay Kit= | =Quant-iT(TM) ssDNA Assay Kit= | ||
==Materials== | ==Materials== | ||
*Quan-iT ssDNA Assay Kit (cat. # Q32851, containing: | |||
*Quant-iT ssDNA reagent, solution in DMSO | *Quant-iT ssDNA reagent, 200x solution in DMSO | ||
* | *Quant-iT buffer | ||
*Oligonucleotide standard ( | *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. | Table 1. Solution | ||
{| border="1" | {| border="1" | ||
! | !Standard/Sample Input | ||
!Volume (ul) of standard/sample | |||
!Volume (ul) of | |||
!Volume (ul) of diluted Working Solution | !Volume (ul) of diluted Working Solution | ||
!Final Oligomer Conc. | !Final Oligomer Conc. | ||
|- | |- | ||
| | |Standards 1/2 | ||
|0 | |10 | ||
| | |190 | ||
| | |0ng/ul | ||
| | |- | ||
|Sample #1-6ab | |||
|4 | |||
|194 | |||
|??? | |||
|- | |||
|} | |||
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. | |||
# Turn off Qubit Fluorometer after last reading: press HOME, scroll to turn off, then press GO to turn it off. | |||
==Results== | |||
{| {{table}} border = "1" | |||
| align="center" style="background:#f0f0f0;"|'''Quant-iT ssDNA''' | |||
| align="center" style="background:#f0f0f0;"| | |||
| align="center" style="background:#f0f0f0;"| | |||
| align="center" style="background:#f0f0f0;"| | |||
| align="center" style="background:#f0f0f0;"| | |||
| align="center" style="background:#f0f0f0;"|'''Orig''' | |||
| align="center" style="background:#f0f0f0;"|'''Orig''' | |||
| align="center" style="background:#f0f0f0;"| | |||
|- | |||
| ||Qubit Reading(ng/mL)||uL used||Dilution||Sample (ng/mL)||Sample Dilution||Sample (ng/uL)||Average (ng/ul) | |||
|- | |||
| 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 | ||
| | |- | ||
|0 | | 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 | |||
|- | |- | ||
|} | |} | ||
== | |||
Purification table | |||
{| {{table}} border = "1" | |||
| align="center" style="background:#f0f0f0;"| | |||
| align="center" style="background:#f0f0f0;"|'''Step''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Purity''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''ssDNA (ng)''' | |||
| align="center" style="background:#f0f0f0;"|'''% Recovery''' | |||
|- | |||
| 1||After first EtOH ppt||1020||9.41||103||105060|| | |||
|- | |||
| 2||After lambda exon||1103||0.92||55.25||60941||58.01% | |||
|- | |||
| 3||After qiaquick||420||2.63||83.42||35035||57.49% | |||
|- | |||
| 4||After USER &DpnII||596||0.7||50.9||26522(*)||75.70% | |||
|- | |||
| 5||After size selection & Nanosep||4047||0.13||0.33||1315||4.96% | |||
|- | |||
| 6||After final EtOH ppt||20||202.350||23.48||470||35.70% | |||
|- | |||
|} | |||
# Purification factor: previous step volume/current step volume | |||
# % Recovery: previous step mass/current step mass | |||
# (*)Subtracted mass of oligo added: 100nM*4238g/moles*1/1000*3ul/rxn*9rxns = 3800ng |
Latest revision as of 22:34, 7 December 2009
Quant-iT(TM) ssDNA Assay Kit[edit]
Materials[edit]
- 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[edit]
- 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. |
---|---|---|---|
Standards 1/2 | 10 | 190 | 0ng/ul |
Sample #1-6ab | 4 | 194 | ??? |
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[edit]
- 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.
- Turn off Qubit Fluorometer after last reading: press HOME, scroll to turn off, then press GO to turn it off.
Results[edit]
Quant-iT ssDNA | Orig | Orig | |||||
Qubit Reading(ng/mL) | uL used | Dilution | Sample (ng/mL) | Sample Dilution | Sample (ng/uL) | Average (ng/ul) | |
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 |
Purification table
Step | Volume (ul) | Purity | Conc. (ng/ul) | ssDNA (ng) | % Recovery | |
1 | After first EtOH ppt | 1020 | 9.41 | 103 | 105060 | |
2 | After lambda exon | 1103 | 0.92 | 55.25 | 60941 | 58.01% |
3 | After qiaquick | 420 | 2.63 | 83.42 | 35035 | 57.49% |
4 | After USER &DpnII | 596 | 0.7 | 50.9 | 26522(*) | 75.70% |
5 | After size selection & Nanosep | 4047 | 0.13 | 0.33 | 1315 | 4.96% |
6 | After final EtOH ppt | 20 | 202.350 | 23.48 | 470 | 35.70% |
- Purification factor: previous step volume/current step volume
- % Recovery: previous step mass/current step mass
- (*)Subtracted mass of oligo added: 100nM*4238g/moles*1/1000*3ul/rxn*9rxns = 3800ng