Klapperich Lab:Notebook/Lab Meeting Notes/2009/11/17: Difference between revisions

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'''‡ Announcements'''<br>
'''‡ Announcements'''<br>
*  Oakridge deadline is 1 Feb 2010. <br>
*  Oakridge deadline is 1 Feb 2010. <br>
* '''PAPERS Drafted before MicroTAS''' Are we done with this? 1st draft by Thur <br>
* '''PAPERS Drafted before MicroTAS''' <br>
 
<br>
<br>
'''‡ Flu R01:Integration''' <br>
'''‡ Flu R01:Integration''' <br>
* New design - address evaporation loss, Reagent delivery and storage solution. Consider marginating flows? <br>
* '''Set up new INTEGRATION MEETING for before 11/25.''' <br>
* New design sent to make Rubber mold  <br>
 
† HDA (Lead: Jaephil, Team:Sonali)<br>
* Start planning R01 for Submission on 2/5/10. MM, JD, CMK. <br>
* HDA chip fabrication training will be after new process settled (comeback from mTAS). <br>
* Two methods, Hot embossing and cutter plotting will be combined to reduce whole process time.<br>
* Dry reagent storage for RT and HDA <br>
* Set-up meeting for 2nd HDA design - Target (Flu or Bacteria), include RT or not, Heat source, address evaporation loss, Reagent delivery and storage solution. Consider marginating flows and include RT? <br>
* Paper resubmission. <br>
 
<br>
<br>
† Sample Concentration (Lead: Jane, Team: Jaephil) <br>
† Sample Concentration (Lead: Jane, Team: Jaephil) <br>
* MDCK Cells are not propagating. Try troubleshooting efforts. Sonali will do on Monday. <br>
* New design sent to make Rubber mold: outline octagon problem. Revised design sent yesterday. <br>
* Cassidy needs to see plaque assay again - so when cells are up, she needs training. <br>
<br>
* Up to high 10^7 for positive control. Silver substrate no signal. <br>
* Jane will train Cassidy once more on Wednesday and Thursday this week. <br>
* Jane working on the cell lysate control. <br>
** MM 040808 A/PR/8/34 flu stock with PFU = 2X10^4 available to Jane to train Cassidy in ultracentrifuging all tubes <br>
* Main loss is at the outlet/sample collection: tangential filtration design in drawing stage. <br>
** of virus, pour out sucrose,pipette remaining with 20uL pipette tip and pool for plaque assay for supplying virus <br>
* Contacted EC Shaw about rubber stamp mold for new design for evaporation. <br>
* Jane working on the cell lysate control. - have many tubes now, done <br>
* Set up COMSOL and StarCD, look for governing equations for simulation of evaporation. <br>
* Set up COMSOL and StarCD, look for governing equations for simulation of evaporation. - ongoing <br>
* Committee meeting setup <br>
<br>
<br>
<br>


† SPE Column Optimization for RNA. (Lead: Sonali, Team: Hussam, Jessie, Brendan) <br>
† SPE Column Optimization for RNA. (Lead: Sonali, Team: Hussam, Jessie, Brendan) <br>
*pH the sample. Optimum 6.6. Make sure that we are there. <br>
* Gold standard Qiagen Kit.?? <br>
* silica nanoparticles from Fluka. <br>
* Report Results today on 2 color dyes, best channel size for 1X 700 nm silica DNA, RNA and 3X 700 nm silica DNA, RNA <br>
* Did adhesive tape instead of bonding help with 3X chips?
* Abandon 3X experiments with current channel designs. If 3X data are encouraging, develop a new channel design for 3X <br>
* What are the next experiments? If types of silica -- Hussam and Jessie describe types today and order by Th 11/19/09 <br>
<br>
*Video with two color dyes and [red(primer) then blue, green, water+tween]
*Video with two color dyes and [red(primer) then blue, green, water+tween]
*repeat experiment reported on 10/27 with 700nm Silica 3X.
*repeat experiment reported on 10/27 with 700nm Silica 3X.
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*PLanning net silica per channel experiments. with 700nm, 3x possible. Deliver around 11/5/09.<br>
*PLanning net silica per channel experiments. with 700nm, 3x possible. Deliver around 11/5/09.<br>
<br>
<br>
* New virus prep (JC, 6/11/09), getting Ct's of 27 for 1:16 dilution and 28 for 1:64 dilution.  Use more diluted sample for future optimization experiments? Channels were 700nm silica/1X. pfu/ml is 10^6. <br>
-Jessie<br>
* Tried 2 more chips with 3X 0.15um SilicaChips are breaking b/c of no crosslinking. SPE was coming out even at 2ml/hr flow rate.<br>
* 3X silica sealing with adhesive on 4 different width channel:  Tried four, no success.  SPE washout, crack forming within the SPE.<br>  
* Running no silica and no SPE chips with virus. (JC/RNA) - No Silica channels grab comparable amounts of RNA with the channels with silica, lower with no SPE.<br>
* Noticed some amounts of SPE coming out even on the channels that seemed to work in the beginning, so probably not retaining 3X silica.<br>
* PCR is running on the samples from the new channel design.  Will compare results with Hussam after the meeting and report back if significantly different.<br>
* Would like to do experiments with different volumes of wash buffer, different flow rates, is it ok?<br>
<br>
<br>
† PCR - CMI  (Lead: Qingqing) <br>
† PCR - CMI  (Lead: Qingqing) <br>
* QQ will work on the initial integration steps of SPE + RT (reservoir)+ PCR. <br>  
* QQ will work on the initial integration steps of SPE + RT (reservoir)+ PCR. <br>  
   - third design was tested with water,big bubbles observed, but the water still could be collected at the outlet.
   - Fourth design was made and tested with PCR reagent(Flu assay).ATCC RNA from Jessie was used.
  - third design was tested with PCR reagent(Flu assay). On-chip PCR does not work.
   - it works!
   - the same experiment was repeated with the reduced hot-start time(five minutes). the on-chip PCR still does not work.
   - patient sample will be tested next.
   * New channel has been designed.
    - molds(SU-8,PDMS,Epox)has been made.
    - chip has been made and tested with water. Both thermal and fluidic control are fine. it will be tested with PCR reagent this week.
    
    
* PCR of C.Difficile DNA
* PCR of C.Difficile DNA
   - primer for toxin A.
   - primer for toxin A.
     - on chip PCR with the optimized primer and template concentration   does not work.
    - product size and concentration was verified by bio-analyzer.
     - MgCl2 concentration was optimized to improve the PCR efficiency.
     - on chip PCR was optimized with higher BSA concentration(0.02%).It did not work.
    - on chip PCR with the optimized MgCl2 concentration does not work.
     - 40 cycle chip and 0.03% (BSA concentration) will be tested.
  - Primer for toxin B.
  - primer for toxin B.  
     New primer sequence from Lisa has been ordered, will be tested when they come.
    -Both two pairs of primers for toxin B works, with a ct value around 13.
     -on chip PCR has been done, the result has not been tested by bio-analyzer.


* PCR2 Paper formatted for LOAC this week. <br>
* PCR2 Paper formatted for LOAC this week. <br>
   need to get back from Cathie
   need to get back from Cathie
* CMK: PCR 1 draft. Analytical Chem. MCK is running more simulations. <br>
* CMK: PCR 1 draft. Analytical Chem. MCK is running more simulations. <br>
<br>
† HDA (Lead: Jaephil, Team:Sonali)<br>
* Start planning R01 for Submission on 2/5/10. MM, JD, CMK. <br>
* HDA chip fabrication training will be after new process settled (comeback from mTAS). <br>
* Two methods, Hot embossing and cutter plotting will be combined to reduce whole process time. New mold design sent. <br>
* Paper submitted 10/6. <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>
* Sonali to train Lisa on SPE. <br>
* Sonali to train Lisa on SPE. <br>
** They don't have syringe pumps and have not asked for training yet. I offered training here which was declined. <br>
* QQ to run test PCR on chip with genomic DNA and Toxin B primers.  <br>
* QQ to run test PCR on chip with genomic DNA and Toxin B primers.  <br>
<br>
<br>
'''‡ Coulter Flu Fraunhofer Project (Lead: Sonali, Team: Sonali, Jessie, Cathie, CMI Folks, Qingqing)'''  
'''‡ Coulter Flu Fraunhofer Project (Lead: Sonali, Team: Sonali, Jessie, Cathie, CMI Folks, Qingqing)'''  
* Meeting 9am Monday (every other week) <br>
* Meeting 9am Monday (every other week) <br>
** Data on AgPath vs Invitrogen one step RT-PCR with plasmid copies and chip extracted RNA on Mon by JC <br>
<br>
<br>
'''‡ Agilent Automated Sample Preparation (Lead: Alex)'''<br>
'''‡ Agilent Automated Sample Preparation (Lead: Alex)'''<br>
* Bacillus subtilis gDNA and cells were tested on SNAP2 machine. gDNA gave good results, pcr signals from cell-samples still low. More tests ongoing. Hot Dog samples will follow. <br>
* So far tested on SNAP2: <br>
* First Draft of Yeast Paper send to Cathie and Alexis. <br>
** B. subtilis gDNA <br>
* Nano-C: which cells shall we use for lysing experiments? <br>
** B. subtilis cells <br>
 
* Planned tests SNAP2: <br>
** B. subtilis cells (higher concentrations) <br>
** MDCK gDNA <br>
** HotDog Sample <br>
*NANO-C: Straw making with two plugs in progress, first lysis test with B.Subtillis cells planned for this week.<br>
*PATH: straw holder for Blackbird is machined  <br>




'''‡ COBRA (Lead: Jaephil, Team: Cathie, Jane for virus only, PHO folks)<br>'''  
'''‡ COBRA (Lead: Jaephil, Team: Cathie, Jane for virus only, PHO folks)<br>'''  
* Metal piece modification for the integration - Fix did not turned out good. Make new or try fix again?<br>
* New metal piece design set.<br>
* paper 1, 1st draft by Thu(10.29): Evap with Sol Gel substrate.  Not integrated. MSSA, E coli. <br>
* Evap paper 1st draft by the end of this week <br>
<br>
<br>
'''‡ Biointerfaces group (Lead: MinCheol, Team: Cathie, MCK, Wong and Meller folks)'''<br>
'''‡ Biointerfaces group (Lead: MinCheol, Team: Cathie, MCK, Wong and Meller folks)'''<br>
Line 101: Line 117:
* Frank: Unfortunately MIA due to interviews.  14-day unstabilized storage PCR results to be analyzed very soon.<br>
* Frank: Unfortunately MIA due to interviews.  14-day unstabilized storage PCR results to be analyzed very soon.<br>
* Frank: PVA arrived. Experiments to commence shortly.<br>
* Frank: PVA arrived. Experiments to commence shortly.<br>
* Sean: Drawings with measurements have been made. Currently working on learning CAD software and making a professional CAD drawing. <br>
* Sean: Design has been submitted to Alex and awaiting the product. I can then get back to working on the optimization of the straws, ie the best monolith composition for extracting DNA/RNA onto the straws <br>
* Sean met with Alex for an integration of straws on black machine. They sketched a design. Sean will make a professional drawing and create a required parts list. Alex will supervise the design. Before handing in the job to the shop floor, the design will be presented to Cathie. <br>
* Sean met with Alex for an integration of straws on black machine. They sketched a design. Sean will make a professional drawing and create a required parts list. Alex will supervise the design. Before handing in the job to the shop floor, the design will be presented to Cathie. <br>
* Mark to begin initial DNA testing with shorter time periods (1,3 days, etc.) to be completed next week.  PCR training to be completed with Hussam. <br>
* Mark to begin initial DNA testing with shorter time periods (1,3 days, etc.) to be completed this(?) week.  PCR training completed. Ready to begin testing.  Waiting for new straw fixture to be ready for testing (where will it be stationed?). <br>





Revision as of 14:18, 17 November 2009

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17 November 2009 Lab Meeting

  • Attending:
  • Missing:

‡ Announcements

  • Oakridge deadline is 1 Feb 2010.
  • PAPERS Drafted before MicroTAS


‡ Flu R01:Integration

  • Set up new INTEGRATION MEETING for before 11/25.

† HDA (Lead: Jaephil, Team:Sonali)

  • Start planning R01 for Submission on 2/5/10. MM, JD, CMK.
  • HDA chip fabrication training will be after new process settled (comeback from mTAS).
  • Two methods, Hot embossing and cutter plotting will be combined to reduce whole process time.
  • Dry reagent storage for RT and HDA
  • Set-up meeting for 2nd HDA design - Target (Flu or Bacteria), include RT or not, Heat source, address evaporation loss, Reagent delivery and storage solution. Consider marginating flows and include RT?
  • Paper resubmission.


† Sample Concentration (Lead: Jane, Team: Jaephil)

  • New design sent to make Rubber mold: outline octagon problem. Revised design sent yesterday.


  • Jane will train Cassidy once more on Wednesday and Thursday this week.
    • MM 040808 A/PR/8/34 flu stock with PFU = 2X10^4 available to Jane to train Cassidy in ultracentrifuging all tubes
    • of virus, pour out sucrose,pipette remaining with 20uL pipette tip and pool for plaque assay for supplying virus
  • Jane working on the cell lysate control. - have many tubes now, done
  • Set up COMSOL and StarCD, look for governing equations for simulation of evaporation. - ongoing


† SPE Column Optimization for RNA. (Lead: Sonali, Team: Hussam, Jessie, Brendan)

  • pH the sample. Optimum 6.6. Make sure that we are there.
  • Gold standard Qiagen Kit.??
  • silica nanoparticles from Fluka.
  • Report Results today on 2 color dyes, best channel size for 1X 700 nm silica DNA, RNA and 3X 700 nm silica DNA, RNA
  • Did adhesive tape instead of bonding help with 3X chips?
  • Abandon 3X experiments with current channel designs. If 3X data are encouraging, develop a new channel design for 3X
  • What are the next experiments? If types of silica -- Hussam and Jessie describe types today and order by Th 11/19/09


  • Video with two color dyes and [red(primer) then blue, green, water+tween]
  • repeat experiment reported on 10/27 with 700nm Silica 3X.
  • controls for these: empties, non-silica monolith and full silica monolith, std. recipe.
  • Load DNA in a straw of 5,50,100ul SPE, quick test of hypothesis: more SPE = more DNA, To be repeated with Alex and Mark
  • Look into modifying Alex's pneumatic setup for the PATH guys. Instead of building the straw array over here NOTE: Sean
  • PLanning net silica per channel experiments. with 700nm, 3x possible. Deliver around 11/5/09.


-Jessie

  • 3X silica sealing with adhesive on 4 different width channel: Tried four, no success. SPE washout, crack forming within the SPE.
  • Noticed some amounts of SPE coming out even on the channels that seemed to work in the beginning, so probably not retaining 3X silica.
  • Would like to do experiments with different volumes of wash buffer, different flow rates, is it ok?


† PCR - CMI (Lead: Qingqing)

  • QQ will work on the initial integration steps of SPE + RT (reservoir)+ PCR.
 - Fourth design was made and tested with PCR reagent(Flu assay).ATCC RNA from Jessie was used.
 - it works!
 - patient sample will be tested next.
 
  • PCR of C.Difficile DNA
 - primer for toxin A.
    - product size and concentration was verified by bio-analyzer.
    - on chip PCR was optimized with higher BSA concentration(0.02%).It did not work.
    - 40 cycle chip and 0.03% (BSA concentration) will be tested.
 - primer for toxin B.   
   -Both two pairs of primers for toxin B works, with a ct value around 13.
   -on chip PCR has been done, the result has not been tested by bio-analyzer.
  • PCR2 Paper formatted for LOAC this week.
 need to get back from Cathie
  • CMK: PCR 1 draft. Analytical Chem. MCK is running more simulations.


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

  • Sonali to train Lisa on SPE.
    • They don't have syringe pumps and have not asked for training yet. I offered training here which was declined.
  • QQ to run test PCR on chip with genomic DNA and Toxin B primers.


‡ Coulter Flu Fraunhofer Project (Lead: Sonali, Team: Sonali, Jessie, Cathie, CMI Folks, Qingqing)

  • Meeting 9am Monday (every other week)
    • Data on AgPath vs Invitrogen one step RT-PCR with plasmid copies and chip extracted RNA on Mon by JC


‡ Agilent Automated Sample Preparation (Lead: Alex)

  • So far tested on SNAP2:
    • B. subtilis gDNA
    • B. subtilis cells
  • Planned tests SNAP2:
    • B. subtilis cells (higher concentrations)
    • MDCK gDNA
    • HotDog Sample
  • NANO-C: Straw making with two plugs in progress, first lysis test with B.Subtillis cells planned for this week.
  • PATH: straw holder for Blackbird is machined


‡ COBRA (Lead: Jaephil, Team: Cathie, Jane for virus only, PHO folks)

  • New metal piece design set.
  • Evap paper 1st draft by the end of this week


‡ Biointerfaces group (Lead: MinCheol, Team: Cathie, MCK, Wong and Meller folks)

  • New experiments happening now.
  • Cathie will submit paper inquiry.


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

  • IRB approved.
  • Cathie submitted the companion BU IRB form for exemption. Revised, still still waiting.


‡ PATH Grant (Lead:Cathie, Team:Frank, Sean, Mark, Jake Trueb (ME), Suma)

  • Phone meeting with PATH this week. 10/13.
  • Senior Project Proposals due in less than one week! (23 Nov.)
  • Frank: Unfortunately MIA due to interviews. 14-day unstabilized storage PCR results to be analyzed very soon.
  • Frank: PVA arrived. Experiments to commence shortly.
  • Sean: Design has been submitted to Alex and awaiting the product. I can then get back to working on the optimization of the straws, ie the best monolith composition for extracting DNA/RNA onto the straws
  • Sean met with Alex for an integration of straws on black machine. They sketched a design. Sean will make a professional drawing and create a required parts list. Alex will supervise the design. Before handing in the job to the shop floor, the design will be presented to Cathie.
  • Mark to begin initial DNA testing with shorter time periods (1,3 days, etc.) to be completed this(?) week. PCR training completed. Ready to begin testing. Waiting for new straw fixture to be ready for testing (where will it be stationed?).