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=25 January 2011 Lab Meeting= | |||
'''‡ Announcements'''<br> | |||
* 11:30-12:30 pm <br> | |||
* REVIEW FOR THE NEW YEAR. | |||
<br> | |||
'''†Trapping Mycobacteria (Jason)'''<br> | |||
* Spent last week in cleanroom, successfully made a new device. | |||
* Cells stay trapped better, as expected (don't have images yet) | |||
'''†Nanomedicine'''<br> | |||
* Sunil <br> | |||
<br> | |||
<br> | |||
'''†Wave 80 project (Andy/Jane/Sam/Sonali)'''<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> | |||
b) Prior to precipitation: +100 ug glycogen, +200mM sodium acetate pH 4.0 (final), +50% isopropanol (final) <br> | |||
<br> | |||
1) Input = 200 uL of either purified RNA / blood + spiked variations / Versant serum + spiked variations <br> | |||
2) Lysis buffer ~ 500 uL total <br> | |||
3) 60 C digestion for 30 min / spin-down for blood only to get rid of particulates<br> | |||
4) Add sodium acetate pH 4.0 (200 mM final) + glycogen (100 ug) <br> | |||
5) Add 700 uL (~ 1 vol) of isopropanol to make it 50% isopropanol final <br> | |||
6) Hard spin-down / 2x wash pellet with 75% EtOH <br> | |||
7) Resuspend pellet in 60 uL water <br> | |||
8) Incubate @ 50C, 10 min for full dissolution <br> | |||
<br> | |||
-- In the context of blood lysis: ------------ <br> | |||
* Working with fresh blood and new lysis buffers, running through straws and testing flow-throughs. (SAB) <br> | |||
* Testing potential filtering method after lysis before introduction to monolith to prevent clogging. (JT/SAB) <br> | |||
* Have ordered Chelex 100 resin from Biorad and see if we can chelate out Fe2+ / Fe3+ before RNA precipitation (SAB / AF) <br> | |||
<br> | |||
-- In the context of Versant / Seracare (serum-only) lysis: ------------ <br> | |||
* 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). | |||
* 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> | |||
* Ran Boom buffer through monolith only straws. Will bDNA the eluent this week.(JZ)<br> | |||
* Process flow throughs. (JZ) <br> | |||
* Chentian - Figuring out a way to do silica channels. First pass: making plug with monolith. <br> | |||
* ABI Taqman for HIV. Waiting for that to arrive. For LTR instead of gag. (MM) <br> | |||
* 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> | |||
* Running RNA storage with stablizer at 0, 1, 4, 12 and 24 hours, qPCR down so analysis on hold. (SAB) <br> | |||
<br> | |||
'''‡ Flu R01:Integration''' <br> | |||
* Paper for chip with Cathie. <br> | |||
need to make an outline <br> | |||
* paper for biostatistics. Thinking phase Sonali.<br> | |||
* Sequence 10 more products - for weekly QC.<br> | |||
done<br> | |||
* Run weekly negative gels. <br> | |||
done<br> | |||
<br> | |||
'''† HDA (Lead:Sonali)'''<br> | |||
*submitted erratum 1/24/11<br> | |||
* <br> | |||
* | |||
<br> | |||
'''† Sample Concentration (Lead: Jane)''' <br> | |||
* Qiagen and SPE done in parallel on original and concentrated samples. <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> | |||
'''† SPE Column Optimization for DNA (Sam and Sonali)''' <br> | |||
* Will do glycogen ppt using DNA as input and titrating amount of pk in lysis buffer. <br> | |||
<br> | |||
'''‡C. diff Project''' (Cathie, Sonali, Satish Singh, Lisa J., His post doc )'''<br> | |||
* Recall: Extracted "ELISA negative samples". They are HDA(toxin A) positive with HDA cloning oligos, but PCR(toxin A) negative with Sara's oligos, and Taqman real-time(toxin B) negative. So right now is in the process of confirming above results and figuring out the reason for this <br> | |||
* Repeat HDA with HDA cloning oligos on different day by using different aliquots of reagents. The result is repeatable. It can also be repeated by doing 2-step HDA reaction <br> | |||
* Clone and Seq. HDA positive samples, and they are true positive <br> | |||
* Optimize phusion enzyme PCR condition for patient samples. Use optimized condition to PCR toxin A gene of more patient samples. Clone & Seq. these amplicons to check whether there is any deletion in toxin A gene of patient samples. <br> | |||
<br> | |||
'''‡ Coulter Flu Fraunhofer Project (Sonali) | |||
* They still need RNA from us. <br> | |||
<br> | |||
'''‡ Biointerfaces<br> | |||
'''* Accepted! <br>''' | |||
<br> | |||
'''‡ CIMIT- Sepsis (Lead:Cathie, Team: Alex)'''<br> | |||
* NEW person needed for this project. <br> | |||
* Negative control of 16s primers as well as of DXS primers (Sonali's Mix) show signals on qPCR. <br> | |||
<br> | |||
'''‡ PATH Grant (Lead:Cathie, Team:Jake, Samantha)<br> | |||
* Straw Extractions on prototypes beginning!!! Two down, four to go before analysis. | |||
* Need to order more straws. Jake is working with Paul on that. <br> | |||
* Working with potential filtering mechanism to prevent straw clogging from whole blood input. <br> | |||
* Begin storage studies using blood as input and spiked RNA. Also will start spiking HIV versant standards into whole blood for storage. <br> | |||
* Extractions on straws using warmed (to 60C) NF water for elution. <br> | |||
Revision as of 08:09, 25 January 2011
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25 January 2011 Lab Meeting‡ Announcements
†Trapping Mycobacteria (Jason)
†Nanomedicine
a) Base formulation: 2.85M Gu-HCl (final)+ 100 ug Proteinase K + 0.015% Tween 20
need to make an outline
done
done
|