Daniel:Notebook/HiResChrPaint/2013-7-12: Difference between revisions
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{| class="wikitable" | {| class="wikitable" | ||
|- align=" | |- align="center" | ||
| '''A''' [[Image:ULS-LabelingRatio.png|400px]]|| [[Image:ULS-ReactionDuration.png|400px]] | | '''A''' || '''B''' | ||
|- | |||
| [[Image:ULS-LabelingRatio.png|400px]]|| [[Image:ULS-ReactionDuration.png|400px]] | |||
|- align="center" | |||
| '''C''' || '''D''' | |||
|- | |- | ||
| [[Image:ULS-ReactionRatio.png|400px]]|| [[Image:ULS-ReactionTemp.png|400px]] | | [[Image:ULS-ReactionRatio.png|400px]]|| [[Image:ULS-ReactionTemp.png|400px]] | ||
|} | |} | ||
Summary: '''A''': Optimal labeling ratio for ULS dyes is around 2%, or 1 in 50 bp. This experiment was done on Alexa 488 so it should be directly applicable to my research. '''B''': Increasing reaction time up to 1 hour should only improve labeling. Optimal time is suggested around 15 minutes but I think 20-30 wouldn't be too bad either. '''C''': Using more dye improves labeling efficiency, rounding off at around 1.4x dye:DNA. This is expected, but might have been an issue if the dye reacted with itself. '''D''': Optimal temperature for the reaction is between 70 and 80 C. Also expected, and not an issue in my protocols. | |||
Basically this tells me that as I expected, adding more dye and incubating longer should get me better yields. | |||
Also of note, reaction temperature is fine and I'm shooting for around 50 bp per dye molecule. The [[Daniel:Protocols/AlexaDyeCouple#ULYSIS|ULYSIS Manual]] | |||
suggests a range of 43 to 67 bases per dye molecule, so this is about right. | |||
So my current thinking is that, as I suggested yesterday, I'm not getting all the dye that I want when I do my labeling reactions. Mabye the dye is sticking to the bottom of the tube or the sides, or in a giant clump. I'm not sure yet. But these results strengthen my suspicions from yesterday that I am not getting uniform dye concentration when I draw samples. | |||
==AmpR 13 and AmpR14== | |||
Continuing AmpR trials. AmpR13 should be a positive control, the 1.5 uL added will finish off the tube. AmpR14 will be the experiment. Going to vortex it thoroughly, and pipette up and down as much as possible. AmpR14 should behave similar to AmpR 13, so long as it is mixed properly. If this fails it will be my last attempt at this for a while, I should think. | |||
*Following [[Daniel:Protocols/AlexaDyeCouple#ULYSIS|ULYSIS protocol]] | |||
1. Add 1 uL DNA in 19 uL labeling buffer (Component C) | |||
2. Denature DNA for 5 minutes at 95C; snap cool on ice | |||
3. Add 1.5 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 | |||
===AmpR13 and AmpR14 Results=== | |||
'''Labeling Efficiency''' | |||
{| 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 20 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" | AmpR13 | |||
| align="center" | 63.5 | |||
| align="center" | 3 | |||
| align="center" | 1.3 | |||
| align="center" | 3.8 | |||
| align="center" | 0.8 | |||
| align="center" | 1.6 | |||
| align="center" | 64 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | AmpR14 | |||
| align="center" | 70.8 | |||
| align="center" | 2.8 | |||
| align="center" | 1.4 | |||
| align="center" | 4.3 | |||
| align="center" | 0.7 | |||
| align="center" | 1.3 | |||
| align="center" | 77 | |||
|} | |||
'''Column Efficiency''' | |||
{| 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="45" | | |||
|style="font-weight:bold" width="65" | Pre ng/uL DNA | |||
|style="font-weight:bold" width="65" | Pre pmol/uL dye | |||
|style="font-weight:bold" width="65" | ng/uL DNA | |||
|style="font-weight:bold" width="65" | pmol/uL dye | |||
|style="font-weight:bold" width="71" | Column Yield (% DNA) | |||
|style="font-weight:bold" width="71" | Column Yield (% dye) | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | AmpR13 | |||
| align="center" | 72.9 | |||
| align="center" | 18.7 | |||
| align="center" | 63.5 | |||
| align="center" | 3 | |||
| align="center" | 75.7 | |||
| align="center" | 14.0 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="15" | AmpR14 | |||
| align="center" | 68.9 | |||
| align="center" | 15 | |||
| align="center" | 70.8 | |||
| align="center" | 2.8 | |||
| align="center" | 89.4 | |||
| align="center" | 16.0 | |||
|} | |||
So it would appear that this trial worked. Since this is the best lead I've gotten, I'm going to go with it. It seems that VIGOROUS vortexing is needed, as well as much pipetting up and down. Note that for this trial I did not pipette the old dye mix (AmpR13) much before adding it to the DNA, but I mixed the DNA quite well for the fresher tube (AmpR14). This is another good sign that I'm on the right track, as the old tube used up the last of the dye and the new tube still has 3 uL left. I guess the next thing is to do a dye couple using the [[Daniel:Notebook/HiResChrPaint/2013-6-10#Nanodrop Results|V6S2 set]]. |
Latest revision as of 22:54, 12 July 2013
S2 Probe Prep (Started 5/29/2013)[edit]
I've been performing some research on the ULS labeling system to see if I can get a good grasp on what's going on before I try labeling again. The most telling, I think, is some figures from a poster I got on Invitrogen's webstite.
Research Notes: ULS[edit]
A | B |
File:ULS-LabelingRatio.png | File:ULS-ReactionDuration.png |
C | D |
File:ULS-ReactionRatio.png | File:ULS-ReactionTemp.png |
Summary: A: Optimal labeling ratio for ULS dyes is around 2%, or 1 in 50 bp. This experiment was done on Alexa 488 so it should be directly applicable to my research. B: Increasing reaction time up to 1 hour should only improve labeling. Optimal time is suggested around 15 minutes but I think 20-30 wouldn't be too bad either. C: Using more dye improves labeling efficiency, rounding off at around 1.4x dye:DNA. This is expected, but might have been an issue if the dye reacted with itself. D: Optimal temperature for the reaction is between 70 and 80 C. Also expected, and not an issue in my protocols.
Basically this tells me that as I expected, adding more dye and incubating longer should get me better yields. Also of note, reaction temperature is fine and I'm shooting for around 50 bp per dye molecule. The ULYSIS Manual suggests a range of 43 to 67 bases per dye molecule, so this is about right.
So my current thinking is that, as I suggested yesterday, I'm not getting all the dye that I want when I do my labeling reactions. Mabye the dye is sticking to the bottom of the tube or the sides, or in a giant clump. I'm not sure yet. But these results strengthen my suspicions from yesterday that I am not getting uniform dye concentration when I draw samples.
AmpR 13 and AmpR14[edit]
Continuing AmpR trials. AmpR13 should be a positive control, the 1.5 uL added will finish off the tube. AmpR14 will be the experiment. Going to vortex it thoroughly, and pipette up and down as much as possible. AmpR14 should behave similar to AmpR 13, so long as it is mixed properly. If this fails it will be my last attempt at this for a while, I should think.
- Following ULYSIS protocol
1. Add 1 uL DNA in 19 uL labeling buffer (Component C) 2. Denature DNA for 5 minutes at 95C; snap cool on ice 3. Add 1.5 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
AmpR13 and AmpR14 Results[edit]
Labeling Efficiency
ng/uL DNA | pmol/uL dye | ug DNA in 20 uL | pmol/uL DNA | dye/probe | dye/100 bp | Base:Dye Ratio (:1) | |
AmpR13 | 63.5 | 3 | 1.3 | 3.8 | 0.8 | 1.6 | 64 |
AmpR14 | 70.8 | 2.8 | 1.4 | 4.3 | 0.7 | 1.3 | 77 |
Column Efficiency
Pre ng/uL DNA | Pre pmol/uL dye | ng/uL DNA | pmol/uL dye | Column Yield (% DNA) | Column Yield (% dye) | |
AmpR13 | 72.9 | 18.7 | 63.5 | 3 | 75.7 | 14.0 |
AmpR14 | 68.9 | 15 | 70.8 | 2.8 | 89.4 | 16.0 |
So it would appear that this trial worked. Since this is the best lead I've gotten, I'm going to go with it. It seems that VIGOROUS vortexing is needed, as well as much pipetting up and down. Note that for this trial I did not pipette the old dye mix (AmpR13) much before adding it to the DNA, but I mixed the DNA quite well for the fresher tube (AmpR14). This is another good sign that I'm on the right track, as the old tube used up the last of the dye and the new tube still has 3 uL left. I guess the next thing is to do a dye couple using the V6S2 set.