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| Line 18: |
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| * Cells stay trapped better, as expected (don't have images yet) | | * Cells stay trapped better, as expected (don't have images yet) |
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| '''†Nanomedicine'''<br> | | '''†Nanomedicine''' (Sunil, Sonali, Cathie and Jen Rosen)<br> |
| * Sunil <br> | | * Sunil <br> |
| <br> | | <br> |
| <br>
| | '''†Wave 80 project (Andy/Sam/Sonali/Cathie)'''<br> |
| '''†Wave 80 project (Andy/Jane/Sam/Sonali)'''<br> | | *Every other week meeting 2-3:30 SKPYE. <br> |
| * The current state of our lysis protocol for RNA precipitation:<br> | |
| --------------------------------------------------------------<br>
| |
| a) Base formulation: 2.85M Gu-HCl (final)+ 100 ug Proteinase K + 0.015% Tween 20 <br>
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| b) Prior to precipitation: +100 ug glycogen, +200mM sodium acetate pH 4.0 (final), +50% isopropanol (final) <br>
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| <br>
| |
| 1) Input = 200 uL of either purified RNA / blood + spiked variations / Versant serum + spiked variations <br>
| |
| 2) Lysis buffer ~ 500 uL total <br>
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| 3) 60 C digestion for 30 min / spin-down for blood only to get rid of particulates<br>
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| 4) Add sodium acetate pH 4.0 (200 mM final) + glycogen (100 ug) <br>
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| 5) Add 700 uL (~ 1 vol) of isopropanol to make it 50% isopropanol final <br>
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| 6) Hard spin-down / 2x wash pellet with 75% EtOH <br>
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| 7) Resuspend pellet in 60 uL water <br>
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| 8) Incubate @ 50C, 10 min for full dissolution <br>
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|
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| <br>
| |
| -- In the context of blood lysis: ------------ <br>
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| * Working with fresh blood and new lysis buffers, running through straws and testing flow-throughs. (SAB) <br>
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| * Testing potential filtering method after lysis before introduction to monolith to prevent clogging. (JT/SAB) <br>
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| * Have ordered Chelex 100 resin from Biorad and see if we can chelate out Fe2+ / Fe3+ before RNA precipitation (SAB / AF) <br>
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| <br>
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| -- In the context of Versant / Seracare (serum-only) lysis: ------------ <br>
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| * Did one round of +SiO2 (pre-acid washed glass beads) test extraction with our Gu-HCl based lysis buffer. Also did one round of silanized SiO2 bead with our Gu-HCl based lysis buffer. Waiting for PCR results (AF).
| |
| <br>
| |
| -- Real-time PCR / Cloning / ssDNA /other stuff ------------ <br>
| |
| * Have ordered Taqman MGB probe for our G100/G25 HIV-gag locus (AF).
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| | |
| * Restreaked clones for exogenous RNA controls where the synthetic RNA template is of non-human, non-HIV origin... the gene segment to-be-cloned actually comes from the cdc6 gene in Xenopus tropicalis (the Western clawed frog). Will digest and find isolate on Wed.(AF).
| |
| * Will try time-course on full pol ssDNA synthesis to minimize E. coli lysis during M13 transfection (AF)<br>
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| * Ran Boom buffer through monolith only straws. Will bDNA the eluent this week.(JZ)<br>
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| * Process flow throughs. (JZ) <br>
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| * Chentian - Figuring out a way to do silica channels. First pass: making plug with monolith. <br>
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| * ABI Taqman for HIV. Waiting for that to arrive. For LTR instead of gag. (MM) <br>
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| * Running bDNA assay on HIV storage in straw at 0, 1, 4, 12 and 24 hours with stablizer, results to come this afternoon. (SAB) <br>
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| * Running RNA storage with stablizer at 0, 1, 4, 12 and 24 hours, qPCR down so analysis on hold. (SAB) <br>
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| <br> | | <br> |
| '''‡ Flu R01:Integration''' <br> | | '''‡ Flu R01:Integration''' <br> |
| * Paper for chip with Cathie. <br> | | * Paper drafting v.8 <br> |
| need to make an outline <br>
| | * Grant renewal will be reviewed on 1/27. <br> |
| * paper for biostatistics. Thinking phase Sonali.<br> | |
| * Sequence 10 more products - for weekly QC.<br>
| |
| done<br>
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| * Run weekly negative gels. <br>
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| done<br>
| |
| <br> | | <br> |
| | |
| '''† HDA (Lead:Sonali)'''<br> | | '''† HDA (Lead:Sonali)'''<br> |
| *submitted erratum 1/24/11<br> | | *submitted erratum 1/24/11.<br> |
| * <br> | | * <br> |
| *
| |
| <br> | | <br> |
| '''† Sample Concentration (Lead: Jane)''' <br> | | '''† Sample Concentration (Lead: Jane, Nga)''' <br> |
| * Qiagen and SPE done in parallel on original and concentrated samples. <br> | | * Meeting Fridays PHO 11-12pm. <br> |
| * 3 new fixtures made. Tried and worked. Need deburring. <br> | | * |
| * New mask and mold made. New chip in progress. <br>
| |
| * Ran exp at Connor lab with current chip and new fixture on fluorescent VSV, plaque assay and imaging results pending. <br>
| |
| <br> | | <br> |
| '''† SPE Column Optimization for DNA (Sam and Sonali)''' <br> | | '''† SERS Franhofer Project (Jared, Chris, Jake, Cathie)''' <br> |
| * Will do glycogen ppt using DNA as input and titrating amount of pk in lysis buffer. <br> | | * Meetings Wed 9-10am at Fraunhofer. <br> |
| <br> | | <br> |
| '''‡C. diff Project''' (Cathie, Sonali, Satish Singh, Lisa J., His post doc )'''<br> | | '''‡C. diff Project''' (Cathie, Sonali, Satish Singh, Lisa J., His post doc )'''<br> |
General Lab Meeting
