Category Archives: probe and plasmid building

RT smears troubleshooting

Posted on January 22, 2014 by admin

A quick look at my history of RT success to ID what started going wrong? Observations not much smearing in early samples. Incorporation rate increases with total amount of mRNA added. Using beads to concentrate mRNA increases production. Using pure … Continue reading →

Posted in probe and plasmid building | Tagged | Comments Off on RT smears troubleshooting

HPLC 12/06/13

Posted on December 6, 2013 by admin

DNA labeling 60 nmol of DNA in 100 uL .1M NaHCO3 (100 mg in 12 mL) .1 – .2 mg dye in 50 uL DMSO (50 uL of 1-2 mg / mL) React at 37C for 1-2 hrs (shake occassionally). … Continue reading →

Posted in probe and plasmid building | Tagged | Comments Off on HPLC 12/06/13

Sunday 09/22/13

Posted on September 22, 2013 by admin

5:30p – 8:30p Bogdan / Rotation project prep: move black IVT reactions to top of upstairs -20C freezer new DNA FISH in situs E6 (E(Pc) region, 15kb) F6 (Y/R 140 kb) Stain E6/F6 originals with 5 uL + 1 uL … Continue reading →

Posted in probe and plasmid building, Summaries | Tagged | Comments Off on Sunday 09/22/13

Notes for deep sequences of libraries

Posted on September 22, 2013 by admin

Bauer Core equipment training option 1 Amplify sublibraries * Buy NEB DNA kit, end repair * blunt end ligation alternative PCR primer gel-extraction Qubit concentration (nanodrop?) [optional] BioAnalyzer at Bauer Core. [optional] qPCR, dilute sample a few times. By Illumina … Continue reading →

Posted in probe and plasmid building | Tagged | Comments Off on Notes for deep sequences of libraries

Wednesday 09/18/13

Posted on September 18, 2013 by admin

9:15a – 11:30p Writing working on BIRS workshop report (9:15a- 12p) Probe making / training with Bogdan test PCR from yesterday common primer concentration was a bit low gel didn’t fully dissolve, might have affected gel running: Repeat PCRs. Repeat … Continue reading →

Posted in probe and plasmid building, Summaries | Tagged , , , | Comments Off on Wednesday 09/18/13

Monday 09/02/13

Posted on September 2, 2013 by admin

8:30a – 6:00p, 8:00p – 11:10p T7 plate reaction planning (?) NEB Quick High Yield T7 template = 10 uL of T7 template (~30-120 ng/uL from spot check) 10 uL NTP buffer mix + 1uL (1/2x) T7 polII mix + … Continue reading →

Posted in probe and plasmid building, Summaries | Tagged , , | Comments Off on Monday 09/02/13

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 →

Posted in probe and plasmid building | Tagged , | Comments Off on RT reactions

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 →

Posted in probe and plasmid building | Tagged , | Comments Off on Library analysis

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 →

Posted in probe and plasmid building, Summaries | Tagged , , | Comments Off on Friday 08/30/13

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 →

Posted in probe and plasmid building | Tagged , | Comments Off on plate PCR for 82 libraries