Revision as of 17:38, 10 July 2013 by >Djacobse
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ULYSIS Dye Coupling (Test 3)[edit]
Based on yesterday's results I think I'm going to see what happens if I use all 5 uL of dye available (see ULYSIS protocol)
in one labeling. I'll use the oligo Noi gave me again, as I have a lot and it doesn't waste probe.
1. Add 5 uL DMSO to Alexa dye 488
2. Add 1 uL DNA in 19 uL labeling buffer (Component C)
3. Denature DNA for 5 minutes at 95C; snap cool on ice
4. Add 5 uL dye to sample
5. Incubate for 15 minutes at 80C, snap cool on ice
6. Purify using Centri-Sep Column
Results[edit]
|
ng/uL DNA
|
pmol/uL dye
|
ug DNA in 20 uL
|
pmol/uL DNA
|
dye/probe
|
dye/100 bp
|
Base:Dye Ratio (:1)
|
AmpR4
|
31.8
|
1.4
|
0.636
|
1.9
|
0.7
|
1.45
|
69
|
BLANK
|
66.6
|
0.2
|
1.332
|
4.0
|
0.0
|
0.10
|
1009
|
This is not far off from the suggested efficiency values (44-67:1). I think that the efficiency can be improved, however,
by also incubating longer.
ULYSIS Dye Coupling (Test 4)[edit]
Based on result from above, I think that using more dye is helpful. However, I also want to see if I can get the same result using less dye
and more time.
1. Add 5 uL DMSO to Alexa dye 488
2. Add 1 uL DNA in 19 uL labeling buffer (Component C) for AmpR5 and AmpR6;
3. Denature DNA for 5 minutes at 95C; snap cool on ice
4. Add 2 uL dye to sample; add 3 uL labeling buffer
5. Incubate AmpR5 for 30 minutes at 80C; incubate AmpR6 for 1 hour, snap cool on ice
6. Purify using Centri-Sep Column
|
ng/uL DNA
|
pmol/uL dye
|
ug DNA in 20 uL
|
pmol/uL DNA
|
dye/probe
|
dye/100 bp
|
Base:Dye Ratio (:1)
|
AmpR5 (30 min incubation)
|
54.5
|
0.5
|
1.1
|
3.3
|
0.2
|
0.3
|
330.3
|
AmpR6 (1 hr incubation)
|
62.6
|
0.4
|
1.3
|
3.8
|
0.1
|
0.2
|
474.2
|
I'm suspicious of these results. It's possible that the extra time completely erradicated the dyes, but I find it more likely that there was
an error in the process. Post-incubation, the samples have had a faint green color that grows stronger with increasing concentration. This
experiment neither showed any hint of a green color, and the pmol/uL values are low enough that I am suspicious they are erroneous.