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==Agi26k0gap Probe Production== ===Production PCR=== *Make Master Mix for 46 well (6 strips) *0 gap first round amplicon -> V6 primers {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn (ul)''' | align="center" style="background:#f0f0f0;"|'''50x rxn (ul)''' |- | [[Matt:LabNotes/2014-5-22#Probe_Expansion_PCR | First round amplicon (10nM)]]||0.2||10 |- | 2x Kapa SYBR MM||50||2500 |- | 100uM pAP1V6U||0.4||20 |- | 100uM AP2V6||0.4||20 |- | H2O||49||2450 |- | Total||100||5000 |- | |} 98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold [[File:2014-07-17_Agi26k0gapProductionPCR.JPG|650px]] ===EtOH Precipitation=== *6 15-ml tubes (with 8 wells of PCR product each) for 0gap **800ul PCR product **2000ul 100% EtOH **2.7ul GlycoBlue **80ul 3M NaOAc pH 5.2-5.5 *Vortexed and put in -80C for overnight *Centrifuged at 3000rpm at 4C for 30min *Discarded supernatant and added 800ul of cold 80% EtOH *Transferred DNA pellet (with 1ml pipette tip) to 6 1.5mL tubes *Centrifuged 1.5ml tubes at 15,000rpm for 5 min on table-top centrifuge *Discarded supernatant and air-dried for 5 min in hood *Resuspended DNA with 100ul H2O ===Qia Column Purification=== *Purified 6 tubes of 100ul in 6 columns following Qiagen protocol *Eluted each column with 40ul and combined all probes into one 1.5ml *Measured concentration with Nanodrop: *~220ul of Agi26k0gap probes: 352.2 ng/ul => 77.5 ug **Every time I tried to measure sample it gave me a warning message that there might be a bubble or unclean pedastal, but consistently measured ~355ng/ul (all 3 times) **In between the 3 sample measurements I cleaned and measured a blank water sample and got +/-2ng/ul with no warning message ***Next day re-measured and got 355.5 ng/ul with no error ===Lambda Exonuclease Digestion=== *Divide into eight pcr tubes of 27.5ul with total amplicon of ~10ug each **Four with old Lambda and Four with new Lambda ***Fourth tube of each set had extra 2.5ul amplicon {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Amplicon||27.5 |- | 10X Lambda Exo Buffer||10 |- | Lambda Exonuclease||10 |- | H2O||52.5 |- | Total||100 |} *Incubated at 37C for 1hr *Purified with of Zymo ssDNA/RNA columns *Eluted with 40ul each *Combined the three tubes with 27.5ul amplicon to measure and compare *Recombined and measured ssDNA with Nanodrop: **New Lambda: 69.5ng/ul x 120ul = 8.34ug **Old Lambda: 65.3ng/ul x 120ul = 7.84ug ***Estimate total yield: ~67ng/ul x 320ul = 21.44ug (21.44/38.75 = 55.3%) *'''Result''': Negligible difference between old and new lambda **Not sure what was causing low yield during Lambda digestion [[Matt:LabNotes/2014-6-24]] **Checked Qiagen vs Zymo columns and that wasn't the reason either ***Whatever it was, things seem to work well now *Continued on: [[Matt:LabNotes/2014-7-22]]
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