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=Probe Prep Test 3= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] Purpose: With the relative success of [[Daniel:Notebook/HiResChrPaint/2013-5-10|previous results]] I am now ready to amplify another probe set. At the same time I want to test the other dye coupling method (ULYSIS), which non-specifically couples with G residues. I will have Alan order the ULYSIS dye and will therefore expand aa-dUTP coupled probes and normal probes. I suspect the normal probes will give me better yields throughout the process. ==Expansion qPCR== 1. Set up master reactions according to table ===aa-dUTP coupled probes=== {| 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" | | width="65" | 20 nM OligoPool | width="65" | 2X Kapa SYBR qPCR MM | width="65" | 100 uM AP1V41U | width="65" | 100 uM AP2V4 | width="65" | 100 uM AP1V61U | width="65" | 100 uM AP2V6 | width="65" | 50 mM aa-dUTP | width="65" | H20 | width="65" | Total Volume |- style="background-color:#8DB4E2;font-size:12pt" align="center" | height="22" | per rxn | align="center" | 0.2 | align="center" | 50 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 0.1 | align="center" | 46 | align="center" | 100 |- style="font-size:12pt" align="center" | height="22" | V4S2 (12.5x) | align="center" | 2.5 | align="center" | 625 | align="center" | 5 | align="center" | 5 | align="center" | 0 | align="center" | 0 | align="center" | 1.5 | align="center" | 611 | align="center" | 1250 |- style="font-size:12pt" align="center" | height="22" | V6S2 (12.5x) | align="center" | 2.5 | align="center" | 625 | align="center" | 0 | align="center" | 0 | align="center" | 5 | align="center" | 5 | align="center" | 1.5 | align="center" | 611 | align="center" | 1250 |} ===Normal Probes (no aa-dUTP)=== {| 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" | | width="65" | 20 nM OligoPool | width="65" | 2X Kapa SYBR qPCR MM | width="65" | 100 uM AP1V41U | width="65" | 100 uM AP2V4 | width="65" | 100 uM AP1V61U | width="65" | 100 uM AP2V6 | width="65" | 50 mM aa-dUTP | width="65" | H20 | width="65" | Total Volume |- style="background-color:#8DB4E2;font-size:12pt" align="center" | height="22" | per rxn | align="center" | 0.2 | align="center" | 50 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 0 | align="center" | 48.2 | align="center" | 100 |- style="font-size:12pt" align="center" | height="22" | V4S2 (12.5x) | align="center" | 2.5 | align="center" | 625 | align="center" | 5 | align="center" | 5 | align="center" | 0 | align="center" | 0 | align="center" | 0 | align="center" | 612.5 | align="center" | 1250 |- style="font-size:12pt" align="center" | height="22" | V6S2 (12.5x) | align="center" | 2.5 | align="center" | 625 | align="center" | 0 | align="center" | 0 | align="center" | 5 | align="center" | 5 | align="center" | 0 | align="center" | 612.5 | align="center" | 1250 |} 2. Aliquot 100 uL into each of 12 qPCR tubes (24 in total for both reactions) 3. PCR protocol: i. PCR 3 min 95C ii. 95C 3 sec iii. 55C 30 sec iv. 72C 20 sec v. plate read vi. goto ii vii. 72C 2 min viii. 16C hold 4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total) 5. [[Daniel:Protocols/EtOHPrecip|Ethanol Precipitation]], added 900 uL 100% EtOH, 30 uL 3 M NaOAc, 1 uL glycoblue 6. Incubated overnight ([[Daniel:Notebook/HiResChrPaint/2013-6-1|continued 6/1/2013]])
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