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=TB12k_Apr2016 V4 Probe Preparation= *[[Matt:LabNotes/2016-5-25 | 10nM Seed Oligos of TB12k_Apr2016 V4]] ===Production PCR=== *Make 2X V4 master mix {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (1X)''' | align="center" style="background:#f0f0f0;"|'''Volume (50X)''' |- | First round amplicon TB12k_Apr2016_V4 (10nM)||0.2||10 |- | 2X KAPA SYBG MM||50||2500 |- | AP1V4U (100uM)||0.4||20 |- | AP2V4 (100uM)||0.4||20 |- | H2O||49||2450 |- | Total||100||5000 |} '''Program''' 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 4C hold *No curve because 100ul reaction volume only works in Biorad PCR machines ===EtOH Precipitation=== *8 15-ml tubes (with 24 wells (3 strips) of PCR product each) for **1,200ul PCR product **3000ul 100% EtOH **4ul GlycoBlue **120ul 3M NaOAc pH 5.2-5.5 *Vortexed and put in -80C for overnight *Centrifuged at 3000rpm at 4C for 30 min *Discarded supernatant and added 800ul of cold 80% EtOH *Transferred DNA pellet (with 1ml pipette tip) to 8 1.5mL tubes *Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C *Discarded supernatant and air-dried for 5 min in hood *Resuspended DNA with 100ul H2O ===Qia Column Purification=== *8 columns elute 50ul each *Nanodrop: 313.6 ng/uL x 400uL = 125.44ug ===Lambda Exo=== *Divide into 8 pcr tubes of 100ul (ideally should be <10ug each) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Amplicon||50 |- | 10X Lambda Exo Buffer||10 |- | Lambda Exonuclease||10 |- | H2O||30 |- | Total||100 |} *Incubated at 37C for 2hr (normally 1hr) *Purified with 8 Zymo ssDNA/RNA columns **Dropped one column so only 7 *Eluted with 40ul each *Recombined and measured ssDNA with Nanodrop: **118.7ng/ul x 280ul = 33.24ug ===Remove Amplification Adapters=== ====USER==== *Split into 5 PCR tubes and add 5ul USER {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | SS-amplicon||56 |- | USER||5 |- | 10X DpnII Buffer||8 |- | H2O||11 |- | Total||80 |} *Incubate at 37C for 2.5 hours ====DpnII==== *Added 15ul of the following {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X DpnII Buffer||2 |- | 100uM RE-DpnII_V4 guide oligo||5 |- | H2O||8 |- |} *Incubated at 94C for 2 min, then 37C for 3 min *Added 5ul DpnII *Incubated at 37C for overnight (~15hrs) ===Zymo Column Purification=== *Eluted 20ul each column (100ul total) *Nanodrop **193.7 ng/ul x 100ul = 19.37 ug (% yield) ===Continued on [[Matt:LabNotes/2016-7-26]]===
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