Monthly Archives: August 2013

Saturday 08/31/13

Posted on August 31, 2013 by admin

8:00a – 5:30p, 10:20p – 10:50p Goals start incorporating feedback from Jeff into review Finish probe making for lib2 test samples. probe making see RT post for results Review feedback Explain thermodynamic models crucial part is that ‘probability of occupancy’ … Continue reading →

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RT reactions

Posted on August 30, 2013 by admin

background prep for RT reactions resuspended common-P1 at 1000 uM concentration 405 primer from Invitrogen not arrived yet compare superscriptII to superscriptIII in yield superscriptII protocol superscriptIII protocol 85 bp 1000 ng/uL ~ 35 pmol per uL = 350 pmol … Continue reading →

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Library analysis

Posted on August 30, 2013 by admin

Labeled version of gel: thoughts small sub-libraries preferentially lost? is this because we only get a fraction of the probes in each sublibrary to amplify? more likely a relative abundance issue — should have substantially more mass in large libraries. … Continue reading →

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Friday 08/30/13

Posted on August 30, 2013 by admin

8:30a-5:00p Goals for today RNA clean up of pilot Lib2 templates. comparison of SuperscriptII and SuperscriptIII RT yields plate scale in vitro transcription? Align multi-day stain get comments on review Coding writing script to aid finding cell matches from Mosaics … Continue reading →

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Thursday 08/29/13

Posted on August 29, 2013 by admin

8:15a – 7:00p Multi-stain analysis try crosslinking 415 beads to fresh, unfixed cells initial beads somewhat clumped and extremely bright, especially under high 405 illumination (or minimal weak 488 illumination). Beads do successfully blead into 647 channel at moderate brightness … Continue reading →

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Wednesday 08/28/13

Posted on August 28, 2013 by admin

8:00a – 7:15p, 10:15p-11:00p Probe making set up in vitro Tx reactions run new gel with ladder: G1 and G7 look blank, others look good (a bit troubling, since G7 nanodrops at ~75ng/uL even though G1 goes blank). Full library … Continue reading →

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plate PCR for 82 libraries

Posted on August 28, 2013 by admin

for each sublib (50 uL reaction) 19 ddH2O 5 uL 5 uM common 1 uL of 1:10 diluted library 25 uL Phusion master 0.25 uL of 200 uM T7 lib specific primer master mix (83x) 1577 uL ddH2O 415 uL … Continue reading →

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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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Monday 08/26/13

Posted on August 26, 2013 by admin

9:45a – 6:30p STORM finish O/N STORM run of AbdA cells Cell culture changed media on 3 new Kc-cell flasks plated media on polyL coated coverglass. cells somewhat sparse, let’s give them some time to grow before fixing. Data clean-up: … Continue reading →

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