Kun:LabNotes/HiResChrPaint/2012-7-12: Difference between revisions
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m (→Dye coupling) |
m (→Dye coupling) |
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| align="center" style="background:#f0f0f0;"|'''Total amount in 2.5ul (ug)''' | | align="center" style="background:#f0f0f0;"|'''Total amount in 2.5ul (ug)''' | ||
| align="center" style="background:#f0f0f0;"|'''Dye''' | | align="center" style="background:#f0f0f0;"|'''Dye''' | ||
| align="center" style="background:#f0f0f0;"|'''Concentration(pmole/ul)''' | |||
|- | |- | ||
| #1-V4||278.49||0.70||Alexa-546 | | #1-V4||278.49||0.70||Alexa-546||7.2 | ||
|- | |- | ||
| #2-V4||432.8||1.08||Alexa-488 | | #2-V4||432.8||1.08||Alexa-488||24.3 | ||
|- | |- | ||
| #3-V4||359.76||0.90||Alexa-594 | | #3-V4||359.76||0.90||Alexa-594||12 | ||
|- | |- | ||
| #4-V4||438.99||1.10||Alexa-647 | | #4-V4||438.99||1.10||Alexa-647||2.0 | ||
|- | |- | ||
| #1-V6||425.3||1.06||Alexa-546 | | #1-V6||425.3||1.06||Alexa-546||7.1 | ||
|- | |- | ||
| #2-V6||427.85||1.07||Alexa-488 | | #2-V6||427.85||1.07||Alexa-488||18.4 | ||
|- | |- | ||
| #3-V6||426.04||1.07||Alexa-594 | | #3-V6||426.04||1.07||Alexa-594||8.6 | ||
|- | |- | ||
| #4-V6||306.88||0.77||Alexa-647 | | #4-V6||306.88||0.77||Alexa-647||2.0 | ||
|- | |- | ||
| | | | ||
|} | |} | ||
*The reaction mixes were incubated at room temperature in dark for 1 hour. | *The reaction mixes were incubated at room temperature in dark for 1 hour. | ||
*The labeled probes were purified individually with Zymo DNA Concentrator-5 columns. | *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. | *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) | |||
Chr.Paint.V4: 177ng/ul | |||
Chr.Paint.V6: 179ng/ul |
Revision as of 00:12, 13 July 2012
Dye coupling
- 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) |
#1-V4 | 278.49 | 0.70 | Alexa-546 | 7.2 |
#2-V4 | 432.8 | 1.08 | Alexa-488 | 24.3 |
#3-V4 | 359.76 | 0.90 | Alexa-594 | 12 |
#4-V4 | 438.99 | 1.10 | Alexa-647 | 2.0 |
#1-V6 | 425.3 | 1.06 | Alexa-546 | 7.1 |
#2-V6 | 427.85 | 1.07 | Alexa-488 | 18.4 |
#3-V6 | 426.04 | 1.07 | Alexa-594 | 8.6 |
#4-V6 | 306.88 | 0.77 | Alexa-647 | 2.0 |
- 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) Chr.Paint.V4: 177ng/ul Chr.Paint.V6: 179ng/ul