Kun:LabNotes/HiResChrPaint/2012-7-12
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Dye coupling[edit]
- Noi did the amplification and prepared single-stranded probes:Noi's lab note.
- 1mL of nuclease free water was added to the Labeling Buffer tube containing sodium bicarbonate. The tube was placed on the Eppendorf thermal shaker at 37C for ~20 min.
- Each vial of dye was resuspended in 2ul DMSO.
- I took 2.5ul of DNA, add 1.5ul of Labeling buffer, then 1ul of dyes. I wasn't able to pipette the exact volume of the dyes due to inexperience and pipetting error.
Probe IDs | Conc. (ng/ul) | Total amount in 2.5ul (ug) | Dye | Concentration(pmole/ul) | Pooled probe conc(ng/ul) | Labeling efficiency | # fluorophores per probe |
#1-V4 | 278.49 | 0.70 | Alexa-546 | 7.2 | 177 | 7.28% | 5.8 |
#2-V4 | 432.8 | 1.08 | Alexa-488 | 24.3 | 177 | 15.81% | 12.6 |
#3-V4 | 359.76 | 0.90 | Alexa-594 | 12 | 177 | 9.39% | 7.5 |
#4-V4 | 438.99 | 1.10 | Alexa-647 | 2 | 177 | 1.28% | 1.0 |
#1-V6 | 425.3 | 1.06 | Alexa-546 | 7.1 | 179 | 4.88% | 3.9 |
#2-V6 | 427.85 | 1.07 | Alexa-488 | 18.4 | 179 | 12.58% | 10.1 |
#3-V6 | 426.04 | 1.07 | Alexa-594 | 8.6 | 179 | 5.90% | 4.7 |
#4-V6 | 306.88 | 0.77 | Alexa-647 | 2 | 179 | 1.91% | 1.5 |
- The reaction mixes were incubated at room temperature in dark for 1 hour.
- Add 5ul 3M NaOAC and 40ul H2O to each tube.
- The labeled probes were purified individually with Zymo DNA Concentrator-5 columns.
- Add 350ul of binding buffer, load the mixture to the column, spin at top speed for 30sec
- Wash twice with 200ul Wash Buffer
- Wash one additional time with 100ul 75% EtOH.
- Eluted with 6ul H2O.
- Four subsets of V4 probes were pooled in one tube, four other subsets for V6 probes were pooled in another tube.
- Measure the yields with Nanodrop (fluorophore-specific concentrations are reported in the table above).
DNA concentration (ng/ul) Amount in 20ul Chr.Paint.V4: 177ng/ul 134 pmole Chr.Paint.V6: 179ng/ul 135 pmole
- Diluted into 2pmole/ul for Chr.Paint.V6
20ul (original tube) + 47.5ul 1XTE = 67.5ul ship 20ul to Brian in Harvard keep 20ul for Scripps Store the rest at 4C (Rui, 4C box)