Revision as of 23:48, 15 April 2013 by >Djacobse
Dye Coupling Test and Gel Quantification
Purpose: Test dye coupling reaction, specifically the purification step, to ensure proper dye-coupled DNA is being captured.
Two samples chosen from Gel Quantification (4/10/13).
Oligos coupled to appropriate dye.
Dye Coupling Reaction
1. Thaw out oligos, sodium bicarbonate solution, DMSO
Use thermo device on Noi's desk to heat DMSO, sodium bicarb to 42 C
2. Add 2.5 uL DNA, 1.5 uL sodium bicarbonate to 0.2 mL eppendorf
3. Denature DNA by incubating at 95 C for 5 minutes, followed by snap cooling on ice
4. Thaw out dyes (they are in big -20C) and resuspend in 2.2 uL DMSO
Use dyes immediately, they do not store
5. Add 1 uL of appropriate dye to oligo set (see below)
Chose V4S2 and V6S2 because of high dna content so I only had to use one dye (Alexa 546)
Probe
|
Alexa Dye
|
V4S1
|
488
|
V4S2
|
546
|
V4S3
|
594
|
V4S4
|
647
|
V6S1
|
488
|
V6S2
|
546
|
V6S3
|
594
|
V6S4
|
647
|
6. Incubate reaction in the dark for 1 hour
7. Column purify using ssDNA kit from Zymo
Urea Gel Quantification
Set up samples according to following table
Sample
|
Low Mass Ladder
|
V4S2 w/Probe
|
V6S2 w/Probe
|
V4S2 w/o Probe
|
V6S2 w/o Probe
|
V6S1 w/o Probe
|
uL sample
|
0.5
|
0.5
|
0.5
|
0.5
|
0.5
|
0.5
|
uL TBE
|
5.5
|
5.5
|
5.5
|
5.5
|
5.5
|
5.5
|
uL 2X buffer
|
6
|
6
|
6
|
6
|
6
|
6
|
Preran gel for ~45 minutes at 250 V
Mixed samples in 0.2 mL eppendorf tubes
Denatured samples for 10 minutes at 65 C immediately before loading
Loaded samples according to following table
Sample
|
Low Mass Ladder
|
V4S2 w/Probe
|
V6S2 w/Probe
|
V4S2 w/o Probe
|
V6S2 w/o Probe
|
V6S1 w/o Probe
|
Lane
|
3
|
4
|
5
|
6
|
7
|
8
|
Ran gel for 20 minutes at 250 V
Added 3 uL of SYBR Gold, incubated for 30 minutes on shaker
Imaged using gel doc
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