Daniel:Notebook/HiResChrPaint/2013-4-15

From ZhangLabWiki
Revision as of 00:26, 16 April 2013 by >Djacobse
Jump to navigation Jump to search

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

Raw xls data

File:ProbeQuant 041513.xls

File:ProbeQuant 041513.jpg


Back to Calendar