Materials and Reagents
1. Labeling Buffer (Sodium Carbonate 1.06g in 100ml DEPC ddH20) PH 8.6 -9.0
2. Dye (Cy dyes from Amersham)
3. QIAquick Nucleotide Removal Kit
Procedure
Note: purifying the probes before the labeling over a QIAquick nucleotide Removal Column gives a much better labeling efficiency, especially for cy3.5 but also for the others.
For cy3.5 only label 5ug instead of 10ug to get better labeling efficiency.
Procedure:
Obtain concentrations of purified probes.
Combine purified probes to10ug total per labeling (for example when using 4 probes to a certain gene, use 2.5ug of each pure probe)
Add 500ul of buffer PN from QIAquick Kit, mix
purify on QIAquick column according to the protocol, but with the following modifications:
collect flow through and apply to column again for extra yield.
Wash with PE buffer 3 times.
!!! elute probes from columns using 40ul H2O, not the elution buffer!!! Otherwise labeling will not work.
Lyophilize probes in a speedvac until dry. Use the speedvac in Meier Rm 323, set for 1 hr, 45°C.
Resuspend the DNA pellet in 10ul of labeling buffer and add it to a tube of dye without touching the dye.
Add another 10ul of labeling buffer to the DNA tube to retrieve any remaining DNA and transfer into the tube of dye without touching the dye
Vortex extensively!!! and spin down the tube of dye and DNA.
Leave in the dark at room temperature (wrapped in foil or in a drawer) overnight to label.
To purify the probes from the free dye, use again the QIAquick columns:
Resuspend labeled probes in 500ul PN and put on column.
spin though columns according to the protocol
take flow-through and put a second time on the column to get best recovery
wash 2x
elute in 100ul H2O
12. Use nanodrop to measure fluorescence using the "microarray" option. Note absorption at A260, Cy3, Cy5.
Data analysis and summary
fill in this table (copy to Excel):
note that in each formula I wrote "1" as line number
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| Sample | Cy Dye | Date | Lot # | Method Purification | Amount Labeled (ug) | Volume of Label (ul) | Dilution Factor | Volume Stock (ul) | A 260 | E 260 | mw (g) | X DNA # n moles of DNA using E | DNA Concentration (ng/ul) using mw | Mass DNA (ug) | A Dye | E Dye | X DYE # n moles of Dye | Ratio | # of Sequences | Total # of Modifiers | # Sequences/ # Modifiers | % Labeling Efficiency using E |
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| * | * | =J1/K1*1000*H1*I1 | =J1*1000*H1*L1/K1 |
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| Cy3 - 150,000 Cy5 - 250000 | =P1/Q1*1000*H1*I1 | =R1/M1 | (number of oligo in mix) | (how many dye molecules/oligo) |
| =R1/M1*V1 |
* - find it on technical datasheet from sigma; calculate average of all oligos.
examples for MBS probe labeled with Cy3 or Cy5:
| MS2 20mer mix labeled by Gal | Cy3 | 14/3/2019 | 9668245 | Qiagen Nucleotide Removal Kit Re-purified | 10.5 | 20 | 1 | 100 | 1.34000 | 201,500 | 6,331 | 0.6650 | 42.10 | 5.12 | 1.81 | 150,000 | 1.206666667 | 1.814502 | 3.00 | 6 | 0.5 | 90.73% |
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| MS2 20mer mix labeled by Gal | Cy5 | 14/3/2019 | 9660231 | Qiagen Nucleotide Removal Kit Re-purified | 10.5 | 20 | 1 | 100 | 1.09000 | 201,500 | 6,331 | 0.5409 | 34.25 | 3.42 | 1.96 | 250,000 | 0.784 | 1.449321 | 3 | 6 | 0.5 | 72.47% |
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