Daniel:Notebook/HiResChrPaint/2013-7-15: Difference between revisions
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6. Add 2 uL dye to sample | 6. Add 2 uL dye to sample | ||
7. Incubate in the dark for 1 hour | 7. Incubate in the dark for 1 hour | ||
8. Add | 8. Add 11 uL nfH20 to sample | ||
9. Purify using [[Daniel:Protocols/CentriSep|Centri Sep]] column | 9. Purify using [[Daniel:Protocols/CentriSep|Centri Sep]] column | ||
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*Following [[Daniel:Protocols/AlexaDyeCouple#ULYSIS|ULYSIS Protocol]] | *Following [[Daniel:Protocols/AlexaDyeCouple#ULYSIS|ULYSIS Protocol]] | ||
1. | 1. Resuspend DNA in 20 uL labeling buffer (Component C) | ||
2. Denature DNA for 5 minutes at 95C; snap cool on ice | 2. Denature DNA for 5 minutes at 95C; snap cool on ice | ||
3. Add 1 uL dye to sample; add 4 uL labeling buffer; AmpR13: Dye from 7/10/13; AmpR14: Dye from 7/11/13 | 3. Add 1 uL dye to sample; add 4 uL labeling buffer; AmpR13: Dye from 7/10/13; AmpR14: Dye from 7/11/13 | ||
4. Incubate for 20 minutes at 80C, snap cool on ice | 4. Incubate for 20 minutes at 80C, snap cool on ice | ||
5. Purify using [[Daniel:Protocols/CentriSep|Centri Sep]] column | 5. Purify using [[Daniel:Protocols/CentriSep|Centri Sep]] column |
Revision as of 19:27, 15 July 2013
S2 Probe Prep (Started 5/29/2013)
ARES Labeling of V6S2
Trying ARES labeling of V6S2 set randomly primed with aa-dUTP.
V6S2 ~ 1.2 ug DNA
- Following ARES Protocol
1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature 2. Resuspend DNA in 2.5uL nfH20 3. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer in PCR tube 4. Denature samples for 5 minutes at 95C, then snap cool using ice box 5. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds *dye cannot be saved for later use. Use immediately! 6. Add 2 uL dye to sample 7. Incubate in the dark for 1 hour 8. Add 11 uL nfH20 to sample 9. Purify using Centri Sep column
ULYSIS Labeling of V6S2
Trying ULS labeling of V6S2 set randomly primed with aa-dUTP.
V6S2 ~ 1.5 ug DNA
- Following ULYSIS Protocol
1. Resuspend DNA in 20 uL labeling buffer (Component C) 2. Denature DNA for 5 minutes at 95C; snap cool on ice 3. Add 1 uL dye to sample; add 4 uL labeling buffer; AmpR13: Dye from 7/10/13; AmpR14: Dye from 7/11/13 4. Incubate for 20 minutes at 80C, snap cool on ice 5. Purify using Centri Sep column