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===Exp.1 eSNPs capture (cont. on the 2007-11-11 exp.)=== Perform PCR on 5ul capturing reactions (100ul for gDNA). x 1 x 12 template: 5ul each ABI 10x buffer: 4.5ul 54ul 25mM MgCl2: 3ul 36ul 10mM dNTP: 1ul 12ul 100uM AmpF2Sol: 0.15ul 1.8ul 100uM AmpRSol: 0.15ul 1.8ul 50X SYBG I: 0.4ul 4.8ul iTaq: 0.5ul 6ul H2O: 35.3ul 430ul 94C 3min -> 10 cycles of (94C 45sec -> 56C 2min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72C 1min) -> 72C 3min -> 15C hold. [[Image:2007-11-14-Hues6-eSNP84k-PCR.png|400px]] Perform PAGE purification. [[Image:2007-11-14-Hues6-seqlib.png | 400px]] Gel quantification: Hues6 gDNA: 3.64ng/ul => 52nM[WRONG] should be 26nM Hues6 cDNA: 1.76ng/ul => 25nM[WRONG] should be 13nM Hues6(no mouse) cDNA: 2.09ng/ul => 30nM[WRONG] should be 15nM Solexa libraries shipped to Gary Gao: <b>First batch, monomer:</b> Hues6gs, 20nM (should be 7nM, corrected before sequencing) Hues6cs, 20nM (should be 7nM, corrected before sequencing) Hues6nms, 20nM (should be 7nM, corrected before sequencing) <b>Second batch, dimer:</b> Hues6gl, 52nM Hues6cl, 25nM Hues6nml, 30nM <b>Hues6gs and Hues6cs was sequenced on 12/05/2007. Here is the summary of the results:</b> <u>Summary of reads:</u> Library # reads # mapped % mappable Hues6cs 14,513,045 7,194,901 49.6% Hues6gs 11,035,520 5,477,493 49.6% # of SNPs with (Fa+Fb)>0.9 and N>20 Hues6gs 14,733 Hues6cs 15,193 Hues6gs&cs 14,318 Genes with >1 heterozygous SNPs: Balanced: 45 (both expressed at the similar level) Allele-specific: 44 (both expressed, one allele is stronger than the other) Mono-allelic: 27 (only one allele is expressed) Conflicting results: 61
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