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==CA12k_Nov2014_V4 Probe Production== *Production of Padlock Probes (V4) [[Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering]] ===Production PCR=== *[[Matt:LabNotes/2015-1-5#Expansion_PCR | 10nM 1st round amplicons]] *V4 master mix made in 2 5ml tubes {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (1X)''' | align="center" style="background:#f0f0f0;"|'''Volume (100X)''' |- | First round amplicon CA12k_Nov14_V4 (10nM)||0.2||20 |- | 2X KAPA SYBG MM||50||5000 |- | AP1V4U (100uM)||0.4||40 |- | AP2V4 (100uM)||0.4||40 |- | H2O||49||4900 |- | Total||100||10000 |} '''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold [[File:20150603_CA12kNov2014_V4_ProductionPCR.JPG|650px]] ===QIAEX-II Purification=== [http://www.qiagen.com/us/resources/download.aspx?id=13d33145-9f64-426a-a43b-394211d8cf2b&lang=en protocol] *Divide into 8 5mL tubes of 1200ul each *Add 3600ul (3X volume) of Buffer QX1 *Vortex the QIAEXII for 30sec *Add 30ul QIAEXII (30ul for every 10ug) *Incubate at 50C while mixing for 10min **Color should be yellow *Centrifuge sample for 30sec *Remove supernatant with a pipette *Wash the pellet with 500ul Buffer QX1 *Resuspend the pellet by vortexing *Transfer to 1.5ml tubes *Centrifuge sample for 30sec and remove supernatant again with pipette *Wash the pellet twice with 500ul Buffer PE *Air-dry the pellet for 30min until the pellet turns white *To elute, resuspend with 20ul H2O and vortexing *Incubate at RT for 5min *Centrifuge for 30sec and pipette out supernatant with sample *Repeat 20ul H2O resuspension and incubation to improve yield 10-15% *Total volume: 300ul *Nanodrop quantification: 155.7 ng/ul -> 46.7 ug ===Lambda Exonuclease Digestion=== *Divide into 6 pcr tubes of 50ul with total amplicon of <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 1hr20min *Purified with 6 Zymo ssDNA/RNA columns *Eluted with 40ul each *Recombined and measured ssDNA with Nanodrop: **59.6ng/ul x 240ul = 14.3ug (61% yield) ===Remove Amplification Adapters=== ====USER==== *Split into 4 PCR tubes and add 5ul USER {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | SS-amplicon||59 |- | USER||5 |- | 10X DpnII Buffer||8 |- | H2O||8 |- | 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 30ul each column (115ul total) *Nanodrop **51.9ng/ul x 115ul = 6.2ug (43% yield) ===PAGE Size Selection=== *Run 2 gels **200V for 40min {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''2X Volume''' |- | V4 Probes||115 |- | TBE-Urea Buffer 2X||115 |} {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''2X Volume''' |- | Low Mass Ladder||2 |- | TBE-Urea Buffer 2X||10 |- | H2O||8 |- | Total||20 |} [[File:2015-06-05_CA12kNov2014_V4_SizeSelect.jpg]] ===EtOH Precipitation=== *Put cut out gel in 4 0.5mL tubes (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT. *Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip. *Added 450 ul of 1X TE buffer *Vortexed for >60min at 37 C in incubator *Centrifuged at 15,000 rpm for 3 min at RT *Transferred the clear spnt. to 4 Nanosep columns and centrifuged at 15,000 rpm for 3 min *Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min *Transferred spnt to 4 fresh 1.5 mL tube (~440ul per tube) *Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2 *Vortexed and placed the 4 tubes at -80C for overnight *Spun 4 tubes at 10,000rpm at 4C for 30min *Discard supernatant and add 650ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min *Discard supernatant and let dry in hood for 10min *Resuspend each tube with 10ul and combine ===Qubit ssDNA Quantification=== 21.4ng/ul => 21.4 ng/ul / (150nt*325/nt + 79Da) = 438nM (60ul)
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