Daniel:Notebook/HiResChrPaint/2013-11-21: Difference between revisions
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>Djacobse (Created page with "=Positive Control 2 Started 11/14/2013 Back to Calendar ==ssNanodrop Results== Following is t...") |
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=Positive Control 2 [[Daniel:Notebook/HiResChrPaint/2013-11-14|Started 11/14/2013]] | =Positive Control 2 ([[Daniel:Notebook/HiResChrPaint/2013-11-14|Started 11/14/2013]])= | ||
[[Daniel:Notebook/HiResChrPaint|Back to Calendar]] | [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] | ||
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|- style="font-size:12pt" align="center" valign="bottom" | |- style="font-size:12pt" align="center" valign="bottom" | ||
|style="font-weight:bold" height="15" | V6S1 | |style="font-weight:bold" height="15" | [[Daniel:Notebook/HiResChrPaint/2013-11-15#ssNanodrop Results|V6S1]] | ||
| align="center" | 103.7 | | align="center" | 103.7 | ||
| align="center" | 2.1 | | align="center" | 2.1 | ||
|- style="font-size:12pt" align="center" valign="bottom" | |- style="font-size:12pt" align="center" valign="bottom" | ||
|style="font-weight:bold" height="15" | V6S2 | |style="font-weight:bold" height="15" | [[Daniel:Notebook/HiResChrPaint/2013-11-15#ssNanodrop Results|V6S2]] | ||
| align="center" | 226.9 | | align="center" | 226.9 | ||
| align="center" | 4.5 | | align="center" | 4.5 | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
|style="font-weight:bold" height="15" | V6S3 | |style="font-weight:bold" height="15" | [[Daniel:Notebook/HiResChrPaint/2013-11-16#ssNanodrop Results|V6S3]] | ||
| align="center" | 144.3 | | align="center" | 144.3 | ||
| align="center" | 2.9 | | align="center" | 2.9 | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
|style="font-weight:bold" height="15" | V6S4 | |style="font-weight:bold" height="15" | [[Daniel:Notebook/HiResChrPaint/2013-11-16#ssNanodrop Results|V6S4]] | ||
| align="center" | 137.2 | | align="center" | 137.2 | ||
| align="center" | 2.7 | | align="center" | 2.7 | ||
|} | |} | ||
==Dye Coupling== | |||
This will be the same as usual dye couplings, except that I'm going to label all dyes with Alexa 488, and I'm going to pool two of the samples together (V4S1,V4S2 and V4S3,V4S4) together before dye coupling, since the labeling is supposed to be for 1-5 ug of DNA, and each of these only has around 2 ug. | |||
===Protocol=== | |||
''For each'', from [[Daniel:Protocols/AlexaDyeCouple#ARES|ARES Protocol]] | |||
0. Dry each sample in vacuum centrifuge, resuspend in 5 uL nfH20 | |||
1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature | |||
2. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer | |||
3. Denature samples for 5 minutes at 95C, then snap cool using ice box | |||
4. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds | |||
*dye cannot be saved for later use. Use immediately! | |||
5. Add 2 uL dye to sample | |||
6. Incubate in the dark for 1 hour | |||
7. Add 15 uL nfH20 to sample | |||
8. [[Daniel:Protocols/CentriSep|Centri Sep column purification]] | |||
===Labeling 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="87" | ng/uL DNA | |||
|style="font-weight:bold" width="65" | pmol/uL dye | |||
|style="font-weight:bold" width="65" | ug DNA in 25 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" | V4S1 | |||
| align="center" | 141.5 | |||
| align="center" | 6.6 | |||
| align="center" | 3.5 | |||
| align="center" | 5.3 | |||
| align="center" | 1.2 | |||
| align="center" | 1.5 | |||
| align="center" | 65 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | V6S3 | |||
| align="center" | 172 | |||
| align="center" | 8.6 | |||
| align="center" | 4.3 | |||
| align="center" | 6.4 | |||
| align="center" | 1.3 | |||
| align="center" | 1.7 | |||
| align="center" | 61 | |||
|} | |||
So everything looks great. Incindentally, I realized that if you want to do a quick calculation through from the nanodrop results (ng/uL DNA and pmol/uL dye), the optimal ratio is close to 20, which will give approximately 60 bp/dye. |
Latest revision as of 16:51, 22 November 2013
Positive Control 2 (Started 11/14/2013)[edit]
ssNanodrop Results[edit]
Following is the single stranded nanodrop results from my 4 probe preps so far.
Sample | ng/uL | ug in 20 uL |
V6S1 | 103.7 | 2.1 |
V6S2 | 226.9 | 4.5 |
V6S3 | 144.3 | 2.9 |
V6S4 | 137.2 | 2.7 |
Dye Coupling[edit]
This will be the same as usual dye couplings, except that I'm going to label all dyes with Alexa 488, and I'm going to pool two of the samples together (V4S1,V4S2 and V4S3,V4S4) together before dye coupling, since the labeling is supposed to be for 1-5 ug of DNA, and each of these only has around 2 ug.
Protocol[edit]
For each, from ARES Protocol
0. Dry each sample in vacuum centrifuge, resuspend in 5 uL nfH20 1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature 2. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer 3. Denature samples for 5 minutes at 95C, then snap cool using ice box 4. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds *dye cannot be saved for later use. Use immediately! 5. Add 2 uL dye to sample 6. Incubate in the dark for 1 hour 7. Add 15 uL nfH20 to sample 8. Centri Sep column purification
Labeling Results[edit]
ng/uL DNA | pmol/uL dye | ug DNA in 25 uL | pmol/uL DNA | dye/probe | dye/100 bp | Base:Dye Ratio (:1) | |
V4S1 | 141.5 | 6.6 | 3.5 | 5.3 | 1.2 | 1.5 | 65 |
V6S3 | 172 | 8.6 | 4.3 | 6.4 | 1.3 | 1.7 | 61 |
So everything looks great. Incindentally, I realized that if you want to do a quick calculation through from the nanodrop results (ng/uL DNA and pmol/uL dye), the optimal ratio is close to 20, which will give approximately 60 bp/dye.