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==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 [[Daniel:Notebook/HiResChrPaint/2013-4-10|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) {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" | width="65" height="16" | Probe | width="65" | Alexa Dye |- style="font-size:12pt" align="center" | height="16" | V4S1 | align="right" | 488 |- style="font-size:12pt" align="center" | height="16" | V4S2 | align="right" | 546 |- style="font-size:12pt" align="center" | height="16" | V4S3 | align="right" | 594 |- style="font-size:12pt" align="center" | height="16" | V4S4 | align="right" | 647 |- style="font-size:12pt" align="center" | height="16" | V6S1 | align="right" | 488 |- style="font-size:12pt" align="center" | height="16" | V6S2 | align="right" | 546 |- style="font-size:12pt" align="center" | height="16" | V6S3 | align="right" | 594 |- style="font-size:12pt" align="center" | height="16" | V6S4 | align="right" | 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 {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" | width="65" height="30" | Sample | width="65" | Low Mass Ladder | width="65" | V4S2 w/Probe | width="65" | V6S2 w/Probe | width="65" | V4S2 w/o Probe | width="65" | V6S2 w/o Probe | width="65" | V6S1 w/o Probe |- style="font-size:12pt" align="center" | height="15" | uL sample | align="center" | 0.5 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 0.5 |- style="font-size:12pt" align="center" | height="15" | uL TBE | align="center" | 5.5 | align="center" | 5.5 | align="center" | 5.5 | align="center" | 5.5 | align="center" | 5.5 | align="center" | 5.5 |- style="font-size:12pt" align="center" | height="15" | uL 2X buffer | align="center" | 6 | align="center" | 6 | align="center" | 6 | align="center" | 6 | align="center" | 6 | align="center" | 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 {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" | width="65" height="30" | Sample | width="65" | Low Mass Ladder | width="65" | V4S2 w/Probe | width="65" | V6S2 w/Probe | width="65" | V4S2 w/o Probe | width="65" | V6S2 w/o Probe | width="65" | V6S1 w/o Probe |- style="font-size:12pt" align="center" valign="bottom" | height="15" | Lane | align="center" | 3 | align="center" | 4 | align="center" | 5 | align="center" | 6 | align="center" | 7 | align="center" | 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 ===Gel Quantification Results=== Raw xls data [[File:ProbeQuant_041513.xls]] [[Image:ProbeQuant_041513.jpg]] {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- align="center" valign="bottom" | width="69" height="12" | Sample | width="69" | V4S2P | width="69" | V6S2P | width="69" | V4S2 | width="69" | V6S2 | width="69" | V6S1 |- align="center" valign="bottom" | height="12" | ng/uL | align="center" | 12.5 | align="center" | 7.1 | align="center" | 32.1 | align="center" | 16.7 | align="center" | 11.2 |} {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" | width="65" height="15" | Sample | width="65" | ng/uL | width="65" | pg/uL | width="65" | pmol/uL | width="65" | uM Probe | width="65" | Probes/oligo |- style="font-size:12pt" align="center" | height="15" | V4S2 | align="center" | 12.5 | align="center" | 12500 | align="center" | 0.5 | align="center" | 1.5 | align="center" | 3.2 |- style="font-size:12pt" align="center" | height="15" | V6S2 | align="center" | 7.1 | align="center" | 7100 | align="center" | 0.3 | align="center" | 0.5 | align="center" | 1.9 |} [[Daniel:Notebook/HiResChrPaint|Back to Calendar]]
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