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= Expansion PCR of new probe set from CustomArrays (90k_oligos_30Sept2014) = * [[noi:DMR220k_LabNotes| '''Link to calendar''']] * 2014-10-15: Received :90k_oligos_30Sept2014 from CustomArray === Oligo info. === * Name: 90k_oligos_30Sept2014 * Conc. 69.89ng/ul, * Volume 80ul in TE buffer. Total amount 5.59ug * [[Kun:LabNotes/MONOD/2014-8-4#Re-design_of_selector_probes| Info from Dr. Zhang's note]] *Sept14 probe set: [[Media:90k_oligos_30Sept2014.txt.gz]] probe set # probes Length Amp primers Chris gDNA_MS_v2 8,045 127bp V6(G*T*CATATCGGTCACTGTU//5Phos/GGGTAGTGTGTATCCTG) Kun cancer_hyb_sept14 51,639 110-130bp V8(T*C*TAATCTAGCGCGACGTCU//5Phos/CCACAAGAGGCGCTATG) Kun LMS_selector 17,342 124-130bp NE(TGCCTAGGACCGGATCAACT/GCTTCGGTTCACGCAATG) Kun padlock_SNPs 12,974 125bp V4(G*A*CTGGAAGAGCACTGTU//5Phos/AGCCTCATGCGTATCCG) Total 90,000 * Length: I would use the average size of the oligo pool to calculate concentration ~125nt = MW=41250g/mole * Conc. : 69.89 ng/ul = 1694.30 (69.89ng/ul/ 41250g/mole) == Expansion PCR (Quick test, round1) == * I initially work on the two subsets, including LMS_selector (NE or eMIP_CA1 primer set) and padlock_SNP (V4 primer set) * I will include the two positive control for the two primer set to confirm that the amplification works fine. Note that I combine F & R primer in final conc. 10uM in the same tube. '''Components Volume (ul) Final conc.''' Seed oligo (1694.3nM) 0.59 100nM F/R primer mix (10uM) 0.40 400nM 2x KAPA SYBG fast MM 5.00 1x H2O 4.01 Total 10.00 * 95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 17-> 72C 3min -> 15C hold [[File:2014-10-15_testExpPCR_Sep14probes.PNG| 500px]] * The qPCR curves of all reactions showed amplification very well ~16 cycles for the new probe set. I then continued to do expansion PCR in larger volume (150ul, 100uM of the template for each subset) without gel verification * <span style="color:red">I SHOULD HAVE VERIFIED AMPLICON SIZE IN TBE GEL BEFORE CONTINUE TO DO EXPANSION PCR IN A LARGE VOLUME EVEN THE qPCR CURVE SHOWED AMPLIFICATION AS USUAL.</span> '''Components Volume (ul)''' Seed oligo (1694.3nM) 8.85 F primer (10uM) 0.60 R primer (10uM) 0.60 2x KAPA SYBG fast MM 75.00 H2O 64.95 Total 150.00 * Split each set in 3X50ul * 95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X '''16'''-> 72C 3min -> 15C hold [[File:2014-10-15_ExpPCR_Sep14probes_LMScluster-padlockSNP.png|500px]] * Purify the 1st amplicons with 2x QIAquick column and elute with 50ul TE buffer * Measure concentration by N.D. * Will add N.D. result. * I then continue to amplify cancer_hyb_sep14 probe set with the same condition of the quick test for the two subsets above for 15 cycles. The amplification worked fine at 15 cycles. [[File:2014-10-15_testExpPCR_Sep14probes_v8.png|500px]] * I ran all 1st round amplicon in 6% TBE gel ** For quick test PCR product, I added 2ul of 6X loading dye to 10ul of PCR product and loaded 3ul in the gel. For column purified amplicons, I loaded 1ul for each [[File:ZhangLab_2 2014-10-16 14hr 04min_test_ExpPCR_Verify.jpg| 400px]] LMS = LMS_selector padl = padlock_SNP PTC = positive control canc = cancer_hyb_sep14 * From the gel image, there is a smear below 100bp band in all the three subsets, which is unexpected. The amplicons size should be ~125-130bp. The sizeof positive control are correct. I am pretty sure that I added the right reagents in each reaction. In addition, the result of expansion PCR in large volume of LMS_selector and padlock_SNP were consistent with the quick test PCR. * I will check the result with Chris for his probe set to see if he has the same issue. ** [[Chris:LabNotes/FateMapping/Calendar/2014/2014-10-16| '''Chris's result''']]<br> * From Chis's result, he got the similar smear below 100bp like the other three subsets amplified by me. * I double-check the primer/adaptor sequences of the designed probes. The design is correct for the four subsets and the number of each subset agrees with Dr. Zhang's note. ** [[Media:2014-10-16-90k-sep14-primer-check.pdf|'''Primer/adaptor sequence check''']] * Dr. Zhang notes that CustomArray has started to synthesize the new batch of these probe sets.
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