Klapperich Lab:Notebook/Lab Meeting Notes/2011/01/25

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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>
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'''†Nanomedicine''' (Sunil, Sonali, Cathie and Jen Rosen)<br>
* Sunil <br>
* Sunil <br>
<br>
<br>
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<br>
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'''†Wave 80 project (Andy/Sam/Sonali/Cathie)'''<br>
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'''†Wave 80 project (Andy/Jane/Sam/Sonali)'''<br>
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*Every other week meeting 2-3:30 SKPYE. <br>
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* The current state of our lysis protocol for RNA precipitation:<br>
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--------------------------------------------------------------<br>
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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>
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1)  Input = 200 uL of either purified RNA / blood + spiked variations / Versant serum + spiked variations <br>
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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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<br>
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--  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).
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<br>
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--  Real-time PCR / Cloning / ssDNA /other stuff ------------ <br>
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* 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).
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* 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>
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* Paper for chip with Cathie. <br>
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* Paper drafting v.8 <br>
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  need to make an outline <br>
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* Grant renewal will be reviewed on 1/27. <br>
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* paper for biostatistics. Thinking phase Sonali.<br>
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* Sequence 10 more  products - for weekly QC.<br>
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  done<br>
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* Run weekly negative gels. <br>
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  done<br>
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<br>
<br>
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'''† HDA (Lead:Sonali)'''<br>
'''† HDA (Lead:Sonali)'''<br>
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*submitted erratum 1/24/11<br>
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*submitted erratum 1/24/11.<br>
* <br>
* <br>
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*
 
<br>
<br>
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'''† Sample Concentration (Lead: Jane)''' <br>
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'''† Sample Concentration (Lead: Jane, Nga)''' <br>
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* Qiagen and SPE done in parallel on original and concentrated samples. <br>
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* Meeting Fridays PHO 11-12pm. <br>
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* 3 new fixtures made. Tried and worked. Need deburring. <br>
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*  
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* New mask and mold made. New chip in progress. <br>
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* Ran exp at Connor lab with current chip and new fixture on fluorescent VSV, plaque assay and imaging results pending. <br>
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<br>
<br>
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'''† SPE Column Optimization for DNA (Sam and Sonali)''' <br>
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'''† SERS Franhofer Project (Jared, Chris, Jake, Cathie)''' <br>
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* Will do glycogen ppt using DNA as input and titrating amount of pk in lysis buffer. <br>
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* 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>

Revision as of 13:12, 25 January 2011

General Lab Meeting Main project page
Previous entry      

25 January 2011 Lab Meeting

‡ Announcements

  • 11:30-12:30 pm
  • REVIEW FOR THE NEW YEAR.


†Trapping Mycobacteria (Jason)

  • Spent last week in cleanroom, successfully made a new device.
  • Cells stay trapped better, as expected (don't have images yet)

†Nanomedicine (Sunil, Sonali, Cathie and Jen Rosen)

  • Sunil


†Wave 80 project (Andy/Sam/Sonali/Cathie)

  • Every other week meeting 2-3:30 SKPYE.


‡ Flu R01:Integration

  • Paper drafting v.8
  • Grant renewal will be reviewed on 1/27.


† HDA (Lead:Sonali)

  • submitted erratum 1/24/11.


† Sample Concentration (Lead: Jane, Nga)

  • Meeting Fridays PHO 11-12pm.


† SERS Franhofer Project (Jared, Chris, Jake, Cathie)

* Meetings Wed 9-10am at Fraunhofer. 


‡C. diff Project (Cathie, Sonali, Satish Singh, Lisa J., His post doc )

  • 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
  • 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
  • Clone and Seq. HDA positive samples, and they are true positive
  • 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.


‡ Coulter Flu Fraunhofer Project (Sonali)

  • They still need RNA from us.


‡ Biointerfaces
* Accepted!

‡ CIMIT- Sepsis (Lead:Cathie, Team: Alex)

  • NEW person needed for this project.
  • Negative control of 16s primers as well as of DXS primers (Sonali's Mix) show signals on qPCR.


‡ PATH Grant (Lead:Cathie, Team:Jake, Samantha)

  • Straw Extractions on prototypes beginning!!! Two down, four to go before analysis.
  • Need to order more straws. Jake is working with Paul on that.
  • Working with potential filtering mechanism to prevent straw clogging from whole blood input.
  • Begin storage studies using blood as input and spiked RNA. Also will start spiking HIV versant standards into whole blood for storage.
  • Extractions on straws using warmed (to 60C) NF water for elution.



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