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==Agi15k_Feb2017_V4 Probe Production== *Production of Padlock Probes (V4) [[Matt:LabNotes/2017-2-8]] ===Notes from [[Matt:LabNotes/2017-4-12|last time]]=== *Do 12 or 13 cycles Production PCR *Experiment with 1hr vs 2hr Lambda digestion *Think of way to maximize USER and DpnII digestion ===Production PCR=== *[[Matt:LabNotes/2017-3-30 | 25nM 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 Agi15k_Feb2017_V4 (25nM)||0.1||10 |- | 2X KAPA SYBG MM||50||5000 |- | AP1V4U (100uM)||0.4||40 |- | AP2V4 (100uM)||0.4||40 |- | H2O||49||4910 |- | Total||100||10000 |} '''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 13 -> 72C 2min -> 15C hold<br> ===EtOH Precipitation=== *8 5-ml tubes (with 12 wells of PCR product each) for V4 **1200ul PCR product **3000ul 100% EtOH **3ul GlycoBlue **120ul 3M NaOAc pH 5.2-5.5 *Vortexed and put in -80C for overnight *Centrifuged at 3,000rpm 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 10 min in hood *Resuspended DNA with 100ul H2O ===Qia Column Purification=== *Purified 8 tubes of 100ul in 8 columns following Qiagen protocol *Eluted each column with 50ul and combined all into one 1.5ml *Measured concentration with Nanodrop: ~400ul of V4 probes: 244.2 ng/ul => ~97.64 ug ===Lambda Exonuclease Digestion=== *Divide into 4 pcr tubes of ~100ul with total amplicon of <10ug each {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Amplicon||96 |- | 10X Lambda Exo Buffer||12 |- | Lambda Exonuclease||12 |- | Total||120 |} *Incubate 2 tubes at 37C for 2hr and 2 tubes at 37C for 1hr *Purified with 8 Zymo ssDNA/RNA columns *Eluted with 40ul each *Recombined and measured ssDNA with Nanodrop: **1hr incubation: 72.3 ng/ul x 160ul = 11.6 ug **2hr incubation: 81.7 ng/ul x 160ul = 13 ug *Conclusion: 2hr incubation is better ===Remove Amplification Adapters=== ====USER==== *Split into 6 PCR tubes and add 5ul USER {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | SS-amplicon||52 |- | USER||5 |- | 10X DpnII Buffer||8 |- | H2O||15 |- | 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 (80ul total) *Nanodrop **125.1ng/ul x 120ul = 15ug (% yield) ===PAGE Size Selection=== *Run 4 gels **200V for 40min {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''4X Volume''' |- | V4 Probes||120 |- | TBE-Urea Buffer 2X||120 |} {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''4X Volume''' |- | Low Mass Ladder||4 |- | TBE-Urea Buffer 2X||20 |- | H2O||16 |- | Total||40 |} ===EtOH Precipitation=== *Put cut out gel in 8 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. **Had to use 2 more 1.5mL tubes to get all gel. Use 2.0mL tubes next time *Added 450 ul of 1X TE buffer to each *Vortexed for 120min at 37 C in incubator *Centrifuged at 15,000 rpm for 3 min at RT *Transferred the clear spnt. to 2 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 fresh 1.5 mL tube *Precipitated in 10 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2 *Vortexed and placed the 10 tubes at -80C overnight *Spun 10 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 8ul and combine ===Qubit Quantification=== *16.7 ng/ul => 16.7 ng/ul / (157nt*325/nt + 79Da) = 326.7 nM (80ul)
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