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=August Library= ===Probes, Samples, and Calculations=== '''Probes:''' Jan09-1: 12.4 ng/ul, 46417 probes; 376 nM Jan09-2: 16.3 ng/ul, 53493 probes; 494 nM Jan09-3: 7.8 ng/ul, 53828 probes; 236 nM '''Samples:''' Sample # Sample name Amount Concentration 1 MH001 1+2 xHeaIII 10 ul 102.7 ng/ul 2 MH010B xMboI 6 ul 183.4 ng/ul 3 MH010B xDpnI 10 ul 100 ng/ul 4 TCF #3 6 ul 200 ng/ul 5 DK4 #2 17 ul 58.9 ng/ul 6 TK4 #2 18 ul 55.9 ng/ul 7 TK9 12 ul 89.4 ng/ul 8 Pol II 10 ul 99.7 ng/ul '''Calculations''' ''Probe: Target'' ratio is ''100:1'' The mass of one genome is 3pg: 200ng of DNA is equal to 200ng/3pg. The amount of probe needed is 200ng/3pg * 250 for each target, and the total amount of probe is (200ng/3pg * 100 * 46417)/(6.0 * 10^23) Example Assume the concentration of probe Jan09-1 to be 10ng/ul. Knowing the length of the probe is ~100 bases and 1 base = 330 Daltons (1bp = 2 bases = 660 Da). Solve: Concentration of Jan09-1 probe: 10ng/ul / 33000 = 303 nM Volume of probe needed= (200ng/3pg * 100 * 46417)/(6.0 * 10^23 * 303 nM) = 1.73ul Volumes needed of each sample: Sample 1 β 200ng / 102.7ng/ul = 1.947ul Sample 2 β 200ng / 183.4ng/ul = 1.091ul Sample 3 β 200ng/ 100.0ng/ul = 2.000ul Sample 4 β 200ng/ 200.0ng/ul = 1.000ul Sample 5 β 200ng/ 58.9ng/ul = 3.396ul Sample 6 β 200ng/ 55.9ng/ul = 3.578ul Sample 7 β 200ng/ 89.4ng/ul = 2.237ul Sample 8 β 200ng/ 99.7ng/ul = 2.006ul ===Reaction Setup=== Sample Number: 1 2 3 4 5 6 7 8 Sample DNA 1.95ul 1.09ul 2.00ul 1.00ul 3.40ul 3.58ul 2.24ul 2.00ul 10x AmpLigase Buffer 2.00ul 2.00ul 2.00ul 2.00ul 2.00ul 2.00ul 2.00ul 2.00ul Jan09-1 1.40ul 1.40ul 1.40ul 1.40ul 1.40ul 1.40ul 1.40ul 1.40ul Jan09-2 1.20ul 1.20ul 1.20ul 1.20ul 1.20ul 1.20ul 1.20ul 1.20ul Jan09-3 2.50ul 2.50ul 2.50ul 2.50ul 2.50ul 2.50ul 2.50ul 2.50ul ddH2O 10.85ul 11.81ul 10.90ul 11.90ul 9.50ul 9.32ul 10.66ul 10.90ul Total Volume/Reaction = 20ul 94C, 2min -> -0.2C/sec to 60C -> 60C, 24h -> add 1ul SLN mix (dNTP 0.5mM, Stoffel 2U/ul, AmpLigase 4U/ul) -> 60C, 24h -> 94C, 1min -> 37C, 1min -> add 2ul Exo I&III -> 37C, 2h -- 90C, 5min -> 4C hold ''' PCR''' (per reaction) Template 10ul iProof Master Mix 50ul 100uM AmpF6.3Sol 0.2ul 100uM AmpR6.3Sol 0.2ul 50x SYBG I 0.2ul ddH2O 40ul 98C, 30sec -> (98C, 10sec -> 60C, 20sec -> 72C, 15sec) x 5 -> (98C, 10sec -> 72C, 20sec) x 20 - > 72C, 3min -> 4C hold
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