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" | &nbsp;
|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" | &nbsp;
|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)

Back to Calendar

ARES Labeling of V6S2

Trying ARES labeling of V6S2 set randomly primed with aa-dUTP.

V6S2 ~ 1.2 ug DNA

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

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.