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=Multi-Color FISH ([[Daniel:Notebook/HiResChrPaint/2014-2-8|Started 02/08/2014]])= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] ==qPCR of BSPP== '''Sample''': *BSPP 1-DMR 220K Exp 1 (10nM); 2/8/11 from Noi #Reaction table {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" |style="background-color:#FFFF00" align="center" width="65" height="45" | |style="font-weight:bold" width="65" | 10 nM OligoPool |style="font-weight:bold" width="65" | 2X Kapa SYBR qPCR MM |style="font-weight:bold" width="65" | 100 uM AP1V61U |style="font-weight:bold" width="65" | 100 uM AP2V6 |style="font-weight:bold" width="65" | 2 mM aa-dUTP |style="font-weight:bold" width="65" | H20 |style="font-weight:bold" width="65" | Total Volume |- style="background-color:#8DB4E2;font-size:12pt" align="center" | height="15" | per rxn | align="center" | 0.4 | align="center" | 50 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 2.5 | align="center" | 46.3 | align="center" | 100 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | BSPP 1 (6.1x) | align="center" | 2.44 | align="center" | 305 | align="center" | 2.44 | align="center" | 2.44 | align="center" | 15.25 | align="center" | 282.43 | align="center" | 610 |} #Aliquot 100 uL into each of 6 qPCR tubes #PCR protocol: ##PCR 3 min 95C ##95C 3 sec ##55C 30 sec ##72C 20 sec ##plate read ##goto ii x17 ##72C 2 min ##16C hold #Pooled 3 samples together each (300 uL each, 2 total) #Column Purification, elute with 40 uL EB #Measure concentrations in nanodrop ===qPCR Results=== '''qPCR Curve''' [[Image:2014-02-21-BSPP1.png|500px]] '''Nanodrop Results''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="65" height="15" | Sample | width="65" | ng/uL | width="65" | ug in 40 uL |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | BSPP1-A | align="center" | 73.9 | align="center" | 3.0 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | BSPP1-B | align="center" | 68.4 | align="center" | 2.7 |} ==Dye Coupling== #Dry samples in vacuum centrifuge #Add 100 uL DMSO to vial containing ULS dye (stable for 6 months at 4C) *For AlexaFluor 488 label using 5 uL DMSO (stable for 1 month at 4C) ***VORTEX THESE SOLUTIONS '''VIGOROUSLY''' BEFORE USE '''EVERY TIME'''! #Resuspend DNA in 21.5 uL labeling buffer (Component C) ##Measure DNA concentration in nanodrop #Denature DNA at 95C for 5 minutes and snap cool on ice; centrifuge briefly #Add appropriate volume of dye to sample (see [[Daniel:Protocols/AlexaDyeCouple#ULYSIS|table 2]]); if necessary add labeling buffer to bring volume to 25 uL #Incubate at 80C for 15 minutes; stop the reaction by plunging the tube into an ice bath #Purify using a [[Daniel:Protocols/CentriSep|Centri Sep column]] '''Label Matrix''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="65" height="15" | Sample | width="73" | Fluorophore | width="65" | ng/uL | width="65" | ug in 20 uL | width="65" | uL dye |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | V6S3 | A-546 | align="center" | 108.4 | align="center" | 2.2 | align="center" | 5 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Cot1-High | A-546 | align="center" | 74 | align="center" | 1.5 | align="center" | 5 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Cot1-Low | A-488 | align="center" | 36.5 | align="center" | 0.7 | align="center" | 1 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | BSPP1-A | A-546 | align="center" | 41.4 | align="center" | 0.8 | align="center" | 5 |} ===Labeling Results=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" | width="65" height="30" | Sample | width="65" | Avg Mol Weight | width="65" | ng/uL DNA | width="65" | pmol/uL dye | width="65" | pmol/uL DNA | width="65" | dye/probe | width="65" | base:dye ratio |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | V6S3 | align="center" | 81 | align="center" | 44.5 | align="center" | 1.6 | align="center" | 1.7 | align="center" | 1.0 | align="center" | 84 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | BSPP1 | align="center" | 100 | align="center" | 67 | align="center" | 1.5 | align="center" | 2.0 | align="center" | 0.7 | align="center" | 135 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Cot1-High | align="center" | 800 | align="center" | 42.4 | align="center" | 3 | align="center" | 0.2 | align="center" | 18.7 | align="center" | 43 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Cot1-Low | align="center" | 100 | align="center" | 28.9 | align="center" | 0.5 | align="center" | 0.9 | align="center" | 0.6 | align="center" | 175 |} [[Category:HRCP]] [[Category:20140208]] [[Category:qPCR]] [[Category:ULYSIS Label]]
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