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:
#Quan-iT ssDNA Assay Kit (cat. # Q32851, containing:
  *Quant-iT ssDNA reagent, solution in DMSO
  *Quant-iT ssDNA reagent, 200x solution in DMSO
  *20xTE (200 mM Tris HCl, 20mM EDTA, pH7.5)
  *Quant-iT buffer
  *Oligonucleotide standard (100ug/ml = 100ng/ul)
*Oligonucleotide standard #1 (0ng/ul in TE Buffer)
#Quan-iT OliGreen Assay tubes (500ul)  
  *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
#Sterile, distilled, DNase free water


Table 1. High range standard curve
==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
{| border="1"
{| border="1"
!
!Standard/Sample Input
!Volume (ul) 1xTE
!Volume (ul) of standard/sample
!Volume (ul) of 2ug/ml Oligomer stock
!Volume (ul) of diluted Working Solution
!Volume (ul) of diluted Working Solution
!Final Oligomer Conc.
!Final Oligomer Conc.
|-
|-
|Standard #1
|Standard #1
|0
|10
|250
|190
|250
|0ng/ul
|1ug/ml
|-
|-
|Standard #2
|Standard #2
|125
|10
|125
|190
|250
|1ng/ul
|500ng/ml
|-
|-
|Standard #3
|Sample #1-6
|225
|1
|25
|199
|250
|???
|100ng/ml
|-
|Standard #4
|247.5
|2.5
|250
|10ng/ml
|-
|Standard #5
|250
|0
|250
|blank
|-
|-
|}
|}
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==
1. Set up 2 tubes for standard and 12 tubes for samples (2x each 1-6)
2. Label tube lids
3. Calculate amount of working solution required.
4. Load in appropriate volumes for each labeled tubes
5. Vortex for 5 s and spin down all tubes
6. Incubate for 2 minutes at room temperature
7. Turn on  Qubit Fluorometer
8. Press  HOME and select Quant-iT ssDNA. Press GO to initiate assay
9. On calibration screen, select Run New Calibration. Press GO
Or select use last calibration
10. Insert standard#1, close lid, press GO
11. Remove standard#1, insert standard#2, close lid, press GO
12. Remove standard#2.
13. Repeat similarly for sample assay tubes.
14. Record readings given by the fluorometer.
==Results==
{| 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
{|-
|}
==Protocol==
==Protocol==
1. Preparations
1. Preparations

Revision as of 01:37, 5 December 2009

Quant-iT(TM) ssDNA Assay Kit

Materials

  1. 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
  1. Sterile, distilled, DNase free water

Starting Notes

  1. Make sure that all solutions are at room temperature
  2. Do not hold the assay tubes in your hand before reading
  3. Do not leave tubes in the fluorometer for longer than necessary as it may warm the assay solution significantly
  4. Do not leave assay solutions exposed to light
  5. Do not overexpose the assay solutions to more than 9 readings as solution is not photostable.
  6. 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:

  1. after 1st EtOH PPT, 1020ul
  2. after Lamda Exonuclease, 1083ul
  3. after QiaQuick, 420ul, diluted 10/3x
  4. after USER & DpnII, 518ul
  5. after size selection & Nanosep, 4047ul
  6. after EtOH PPT, 20ul, diluted to 0.1x

Protocol

1. Set up 2 tubes for standard and 12 tubes for samples (2x each 1-6) 2. Label tube lids 3. Calculate amount of working solution required. 4. Load in appropriate volumes for each labeled tubes 5. Vortex for 5 s and spin down all tubes 6. Incubate for 2 minutes at room temperature 7. Turn on Qubit Fluorometer 8. Press HOME and select Quant-iT ssDNA. Press GO to initiate assay 9. On calibration screen, select Run New Calibration. Press GO

Or select use last calibration

10. Insert standard#1, close lid, press GO 11. Remove standard#1, insert standard#2, close lid, press GO 12. Remove standard#2. 13. Repeat similarly for sample assay tubes. 14. Record readings given by the fluorometer.

Results

{!Sample
{!Conc. (ng/ul)

Protocol

1. Preparations

a. Prepare 1xTE buffer (1 ml per each sample to be assayed)
b. Prepare working solution by 200 fold dilution of OliGreen ssDNA reagent (in DMSO). 
For best results, use working solution within few hours of preparation.
c. Dilute oligonucleotide standard 50 fold to 2ug/ml