Dinh/NOTES/2009-12-4: Difference between revisions

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!Final Oligomer Conc.
!Final Oligomer Conc.
|-
|-
|Standard #1
|Standards 1/2
|10
|10
|190
|190
|0ng/ul
|0ng/ul
|-
|-
|Standard #2
|Sample #1-6ab
|10
|4
|190
|194
|1ng/ul
|-
|Sample #1-6
|1
|199
|???
|???
|-
|-
Line 70: Line 65:
# Repeat similarly for sample assay tubes.
# Repeat similarly for sample assay tubes.
# Record readings given by the fluorometer.
# 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==
==Results==
{| border="1"
{| {{table}} border = "1"
!Sample
| align="center" style="background:#f0f0f0;"|'''Quant-iT ssDNA'''
|1a
| align="center" style="background:#f0f0f0;"|
|1b
| align="center" style="background:#f0f0f0;"|
|2a
| align="center" style="background:#f0f0f0;"|
|2b
| align="center" style="background:#f0f0f0;"|
|3a
| align="center" style="background:#f0f0f0;"|'''Orig'''
|3b
| align="center" style="background:#f0f0f0;"|'''Orig'''
|4a
| align="center" style="background:#f0f0f0;"|
|4b
|-
|5a
|  ||Qubit Reading(ng/mL)||uL used||Dilution||Sample (ng/mL)||Sample Dilution||Sample (ng/uL)||Average (ng/ul)
|5b
|-
|6a
| 1a||209||4||50||10450||10||104.5||
|6b
|-
| 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||
|-
|-
!Conc. (ng/ul)
| 6b||4.6||4||50||230||100||23||23.48
|1a
|1b
|2a
|2b
|3a
|3b
|4a
|4b
|5a
|5b
|6a
|6b
|-
|-
|}
|}
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]

  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.
Standards 1/2 10 190 0ng/ul
Sample #1-6ab 4 194 ???

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[edit]

  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
  1. Insert standard#1, close lid, press GO
  2. Remove standard#1, insert standard#2, close lid, press GO
  3. Remove standard#2.
  4. Repeat similarly for sample assay tubes.
  5. Record readings given by the fluorometer.
  6. 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%
  1. Purification factor: previous step volume/current step volume
  2. % Recovery: previous step mass/current step mass
  3. (*)Subtracted mass of oligo added: 100nM*4238g/moles*1/1000*3ul/rxn*9rxns = 3800ng