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

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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 11:09, 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



†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.



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