Category Archives: probe and plasmid building

RT smears troubleshooting

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

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HPLC 12/06/13

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

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Sunday 09/22/13

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

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Notes for deep sequences of libraries

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

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Wednesday 09/18/13

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

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Monday 09/02/13

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

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RT reactions

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

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

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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plate PCR for 82 libraries

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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