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==Dye coupling== *Noi did the amplification and prepared single-stranded probes:[[Noi/NOTES/2012-5-16|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. {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe IDs''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount in 2.5ul (ug)''' | align="center" style="background:#f0f0f0;"|'''Dye''' | align="center" style="background:#f0f0f0;"|'''Concentration(pmole/ul)''' | align="center" style="background:#f0f0f0;"|'''Pooled probe conc(ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Labeling efficiency''' | align="center" style="background:#f0f0f0;"|'''# 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)
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