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
†Wave 80 project (Andy/Jane/Sam/Sonali)
- The current state of our lysis protocol for RNA precipitation:
a) Base formulation: 2.85M Gu-HCl (final)+ 100 ug Proteinase K + 0.015% Tween 20
b) Prior to precipitation: +100 ug glycogen, +200mM sodium acetate pH 4.0 (final), +50% isopropanol (final)
1) Input = 200 uL of either purified RNA / blood + spiked variations / Versant serum + spiked variations
2) Lysis buffer ~ 500 uL total
3) 60 C digestion for 30 min / spin-down for blood only to get rid of particulates
4) Add sodium acetate pH 4.0 (200 mM final) + glycogen (100 ug)
5) Add 700 uL (~ 1 vol) of isopropanol to make it 50% isopropanol final
6) Hard spin-down / 2x wash pellet with 75% EtOH
7) Resuspend pellet in 60 uL water
8) Incubate @ 50C, 10 min for full dissolution
-- In the context of blood lysis: ------------
- Working with fresh blood and new lysis buffers, running through straws and testing flow-throughs. (SAB)
- Testing potential filtering method after lysis before introduction to monolith to prevent clogging. (JT/SAB)
- Have ordered Chelex 100 resin from Biorad and see if we can chelate out Fe2+ / Fe3+ before RNA precipitation (SAB / AF)
-- In the context of Versant / Seracare (serum-only) lysis: ------------
- 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).
-- Real-time PCR / Cloning / ssDNA /other stuff ------------
- 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)
- Ran Boom buffer through monolith only straws. Will bDNA the eluent this week.(JZ)
- Process flow throughs. (JZ)
- Chentian - Figuring out a way to do silica channels. First pass: making plug with monolith.
- ABI Taqman for HIV. Waiting for that to arrive. For LTR instead of gag. (MM)
- Running bDNA assay on HIV storage in straw at 0, 1, 4, 12 and 24 hours with stablizer, results to come this afternoon. (SAB)
- Running RNA storage with stablizer at 0, 1, 4, 12 and 24 hours, qPCR down so analysis on hold. (SAB)
‡ Flu R01:Integration
- Paper for chip with Cathie.
need to make an outline
- paper for biostatistics. Thinking phase Sonali.
- Sequence 10 more products - for weekly QC.
done
- Run weekly negative gels.
done
† HDA (Lead:Sonali)
- submitted erratum 1/24/11
† Sample Concentration (Lead: Jane)
- Qiagen and SPE done in parallel on original and concentrated samples.
- 3 new fixtures made. Tried and worked. Need deburring.
- New mask and mold made. New chip in progress.
- Ran exp at Connor lab with current chip and new fixture on fluorescent VSV, plaque assay and imaging results pending.
† SPE Column Optimization for DNA (Sam and Sonali)
- Will do glycogen ppt using DNA as input and titrating amount of pk in lysis buffer.
‡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.
|
General Lab Meeting
