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===Expansion PCR=== *[[Matt:LabNotes/2014-12-18]] conclusion: 21 cycles of PCR starting with 100nM seed oligos *Make 2 tubes: **V6 primers - CA12k_Nov2014_Contig_RevComp **V8 primers - CA12k_Nov2014_Const_RevComp {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Contig of Exons RevComp(V6)''' | align="center" style="background:#f0f0f0;"|'''Constitutive Exons RevComp(V8)''' |- | CA12k_Nov2014 Seed Oligos (900nM)||15||15 |- | 2X KAPA SYBG MM||67.5||67.5 |- | 100uM AP1V6U||0.6||0 |- | 100uM AP2V6||0.6||0 |- | 100uM AP1V8U||0||0.6 |- | 100uM AP2V8||0||0.6 |- | H2O||51.3||51.3 |- | Total||135||135 |} '''Program''' 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 21 -> 72C 2min -> 15C hold [[File:012614_CA12kNov14_ExpansionPCR_V6V8.JPG|650px]] *Did not think V6 amplified correctly (possibly due to underpipetting a primer) but [[Matt:LabNotes/2015-1-5#Expansion_PCR|V4]] showed a similar curve **Nanodrop also shows V6 amplified as much or more than V4 *Purified each with 1 Qiagen column and eluted with 50ul H2O *Nanodrop: **V6: 11.8 ng/ul -> 11.8 ng/ul / (188bp*660Da/bp) = 95nM **V8: 16.2 ng/ul -> 16.2 ng/ul / (190bp*660Da/bp) = 129nM *Diluted to 10nM **V6: 48ul x 95nM = '''456ul''' x 10nM ***Add 408ul H2O **V8: 48ul x 129nM = '''619ul''' x 10nM ***Add 571ul H2O ====Gel Check==== *Use 5ul of 10nM expansion PCR'd probes + 5ul 2X Loading dye *Use 2ul 10nM Agi26k0gap expansion PCR'd probes as positive control [[File:2015-01-26_1stRndAmplicon_V6V8_Gel.jpg]] *Apparently Nanodrop is not accurate enough at low conc *V6 is missing a band at 188bp... **Try Expansion PCR with V6 primers on the last of the seed oligos ====V6 Expansion PCR Try 2==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | CA12k_Nov2014 Seed Oligos (900nM)||14 |- | 2X KAPA SYBG MM||50 |- | 100uM AP1V6U||1 |- | 100uM AP2V6||1 |- | H2O||34 |- | Total||100 |} '''Program''' 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 30 -> 72C 2min -> 15C hold [[File:012614_CA12kNov14_ExpansionPCR_V6_try2.JPG|650px]] *Stopped after 22 cycles *Purified each with 1 Qiagen column and eluted with 50ul H2O *Nanodrop: **V6: 17.9 ng/ul -> 17.9 ng/ul / (188bp*660Da/bp) = 144nM *Diluted to 10nM **V6: 48ul x 144nM = '''691ul''' x 10nM ***Add 643ul H2O
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