Daniel:Notebook/HiResChrPaint/2013-7-15: Difference between revisions
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8. Add 11 uL nfH20 to sample | 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 | ||
===ARES Results=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-size:12pt" align="center" | |||
| align="center" width="65" height="30" | | |||
|style="font-weight:bold" width="65" | ng/uL DNA | |||
|style="font-weight:bold" width="65" | pmol/uL dye | |||
|style="font-weight:bold" width="65" | ug DNA in 15 uL | |||
|style="font-weight:bold" width="71" | pmol/uL DNA | |||
|style="font-weight:bold" width="71" | dye/probe | |||
|style="font-weight:bold" width="71" | dye/100 bp | |||
|style="font-weight:bold" width="71" | Base:Dye Ratio (:1) | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | V6S2 | |||
| align="center" | 189.1 | |||
| align="center" | 7.9 | |||
| align="center" | 2.8 | |||
| align="center" | 7.1 | |||
| align="center" | 1.1 | |||
| align="center" | 1.4 | |||
| align="center" | 73 | |||
|} | |||
==ULYSIS Labeling of V6S2== | ==ULYSIS Labeling of V6S2== | ||
Line 35: | Line 60: | ||
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 | ||
===ULYSIS Results=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-size:12pt" align="center" | |||
| align="center" width="65" height="30" | | |||
|style="font-weight:bold" width="65" | ng/uL DNA | |||
|style="font-weight:bold" width="65" | pmol/uL dye | |||
|style="font-weight:bold" width="65" | ug DNA in 15 uL | |||
|style="font-weight:bold" width="71" | pmol/uL DNA | |||
|style="font-weight:bold" width="71" | dye/probe | |||
|style="font-weight:bold" width="71" | dye/100 bp | |||
|style="font-weight:bold" width="71" | Base:Dye Ratio (:1) | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | V6S2 | |||
| align="center" | 237 | |||
| align="center" | 6.1 | |||
| align="center" | 3.6 | |||
| align="center" | 8.9 | |||
| align="center" | 0.7 | |||
| align="center" | 0.8 | |||
| align="center" | 118 | |||
|} | |||
Great results from ARES. Good but not great from ULS. |
Revision as of 19:48, 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
ARES Results
ng/uL DNA | pmol/uL dye | ug DNA in 15 uL | pmol/uL DNA | dye/probe | dye/100 bp | Base:Dye Ratio (:1) | |
V6S2 | 189.1 | 7.9 | 2.8 | 7.1 | 1.1 | 1.4 | 73 |
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 4. Incubate for 20 minutes at 80C, snap cool on ice 5. Purify using Centri Sep column
ULYSIS Results
ng/uL DNA | pmol/uL dye | ug DNA in 15 uL | pmol/uL DNA | dye/probe | dye/100 bp | Base:Dye Ratio (:1) | |
V6S2 | 237 | 6.1 | 3.6 | 8.9 | 0.7 | 0.8 | 118 |
Great results from ARES. Good but not great from ULS.