Category Archives: probe and plasmid building

T7 in vitro Tx

Posted on August 28, 2013 by admin

Library2, samples G1-8 skip sample G1, no DNA recovery NEB For each sub-library, mix the following in a single PCR tube or well of a PCR strip or plate 8 uL DNA template + 2uL ddH2o 1.5 uL 10X T7 … Continue reading →

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PCR

Posted on August 28, 2013 by admin

Amplifying G1-8 using T7 primers for each sublib 18.5 ddH2O 5 uL 5uM common 1 uL library (cut to .5, at 1uL I can only do each lib once) 25 uL Phusion master master mix (9x) 166.5 uL ddH2O 45 … Continue reading →

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Protected: more probe making approaches

Posted on May 14, 2013 by admin

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qubit measurements

Posted on April 17, 2013 by admin

All numbers in ng/uL (equivelantly ug/mL). Original volume is 1 mL so this equivelantly DNA mass. Though some samples were split multiple ways, EtOH is 2:1 with vs without glycogen. Filtration was done with 150 uL of 1 mL PCR. … Continue reading →

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experimental oligopaint PCR 3

Posted on April 14, 2013 by admin

Samples (1 mL scale) y nicked 2x Ya-primer b nicked 1x Ba-primer + 1 x CPPT ubx nicked 1x UBx-adapter-primer + 1x CPPT y PPT: 5 uL YA + 20 uL PPT y PPT control: 5 uL YA + 20 … Continue reading →

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denaturing diagnostic gels

Posted on April 14, 2013 by admin

Gel 1, 5% Agarose, 200 V in sodium-Borax (300 V melts gel). Gel 2, 15% Urea, 120V 2 hrs Gel 3 15% Urea gel, 120 V 50 min in 37C room Gel 4, 10% precast Urea denaturing gel, 120 V, … Continue reading →

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qubit data

Posted on April 12, 2013 by admin

results prior to extraction y prot contr = too high BP 1/2 PCR = 38 ug/mL 7:20 P, reusing standards, 10.9 BP control = 72.4 ug/mL BP = 35 ug/mL Y-T em 1/2 = 9.16 ug/mL. 7:20P, reusing standards, = … Continue reading →

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oligopaint-PCR

Posted on April 4, 2013 by admin

Calculating concentrations (Qubit) and molecule numbers: starting primer = 30 ng in 300 uL in 1 ng/uL, added 100 uL = 100 ng 100 uM = 100E-12 mols/uL add 100 uL, = 10 nmols starting primer. Y PCR: 73.7 ng/mL … Continue reading →

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Wednesday 04/03/13

Posted on April 3, 2013 by admin

9:45 A – 8:00 P, 9:30 P – 12:00 A General Send notes on sog data to Mounia Training on Qbit. Important point: measure samples between 2 and 2.5 minutes after mixing in reaction dye. STORM Analysis Manual analysis of … Continue reading →

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oligopaint probe making planning

Posted on April 1, 2013 by admin

emPCR ready libraries: AbdA AbdB Ubx en-locus 100kb Black 100kb Yellow 100kb Green 100kb Synthesis schemes: Teritaries + Nicking PCR using extended primers = Forward primer (21bp) + Secondary binding site (32 bp) Nick off R primer Denaturing PAGE Gel … Continue reading →

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