Klapperich Lab:Notebook/Lab Meeting Notes/2009/11/24: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
No edit summary
(11 intermediate revisions by 7 users not shown)
Line 20: Line 20:
† HDA (Lead: Jaephil, Team:Sonali)<br>
† HDA (Lead: Jaephil, Team:Sonali)<br>
* Start planning R01 for Submission on 2/5/10. MM, JD, CMK. <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>
* Dry reagent storage for RT and HDA - COP test well array provided fo Sonali <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>
* 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>
* Paper resubmission. Cathie will do Wed. <br>
 
<br>
<br>
† Sample Concentration (Lead: Jane, Team: Jaephil) <br>
† Sample Concentration (Lead: Jane, Team: Jaephil) <br>
* New design sent to make Rubber mold: outline octagon problem. Revised design sent yesterday.  <br>
* New design sent to make Rubber mold: outline octagon problem. Revised design sent yesterday.  <br>
<br>
* Jane will train Cassidy once more on Wednesday and Thursday this week. <br>
* Jane will train Cassidy once more on Wednesday and Thursday this week. <br>
** MM 040808 A/PR/8/34 flu stock with PFU = 2X10^4 available to Jane to train Cassidy in ultracentrifuging all tubes <br>
** MM 040808 A/PR/8/34 flu stock with PFU = 2X10^4 available to Jane to train Cassidy in ultracentrifuging all tubes <br>
Line 36: Line 32:
* Set up COMSOL and StarCD, look for governing equations for simulation of evaporation. - ongoing <br>
* Set up COMSOL and StarCD, look for governing equations for simulation of evaporation. - ongoing <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>
*pH the sample. Optimum 6.6. Make sure that we are there. 3M GUSCN stands at pH 4.2 very acidic (some NaOH)<br>
* Gold standard Qiagen Kit.?? <br>
* "Measure again 1:1 with DEPC water, 1:1 with PBS"
* silica nanoparticles from Fluka. <br>
* Silica nanoparticles from Fluka. 15nm "order"<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>
*Still working on the 150nm particles 1X experiments with lambda. HM<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>
<br>
*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. <br>
* Load DNA in a straw of 5,50,100ul SPE, quick test of hypothesis: more SPE = more DNA,  <i>To be repeated with Alex and Mark </i>  <br>
*'' Look into modifying Alex's pneumatic setup for the PATH guys. Instead of building the straw array over here NOTE: Sean'' <br>
*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>
-Jessie<br>
* 150nm and 700nm silica with 0.5mm (3.6uL SPE) channels.<br>
* 3X silica sealing with adhesive on 4 different width channel:  Tried four, no success. SPE washout, crack forming within the SPE.<br>  
* 150nm Silica did not improve Ct on average, but one channel with 700nm silica performed exceptionally well, which brought the average up.<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>
*"relationship between PFU/ml and copy # of RNA. Do this via a serial dilution of Virus, put through Qiagen kit. Assuming that Qiagen is 100% efficient." <br>  
* Would like to do experiments with different volumes of wash buffer, different flow rates, is it ok?<br>
* "Jessie get trained for real on SEM.*"<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>  
   - Fourth design was made and tested with PCR reagent(Flu assay).ATCC RNA from Jessie was used.
   - SPE+RT+PCR chip is designed.
  - it works!
  - patient sample will be tested next.
    
    
* PCR of C.Difficile DNA
* PCR of C.Difficile DNA<br>
   - primer for toxin A.
   - toxin A.
    - product size and concentration was verified by bio-analyzer.
    HDA assay for toxin A is tested in tube. It works
    - on chip PCR was optimized with higher BSA concentration(0.02%).It did not work.
    HDA chip will be designed, and tested.
    - 40 cycle chip and 0.03% (BSA concentration) will be tested.
    SPE DNA from lisa for test?
   - primer for toxin B.   
   - toxin B.   
     -Both two pairs of primers for toxin B works, with a ct value around 13.
     on chip PCR works.
    -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
Line 85: Line 68:
'''‡ 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>
* "ASB will give action items." <br>
<br>
<br>
'''‡ Agilent Automated Sample Preparation (Lead: Alex)'''<br>
'''‡ Agilent Automated Sample Preparation (Lead: Alex)'''<br>
* So far tested on SNAP2: <br>
* So far tested on SNAP2: <br>
** B. subtilis gDNA <br>
** B. subtilis gDNA <br>
** B. subtilis cells <br>
** B. subtilis cells, high Cts for the range tested. <br>
* Planned tests SNAP2: <br>
* Planned tests SNAP2: <br>
** B. subtilis cells (higher concentrations) <br>
** B. subtilis cells (higher concentrations) <br>
** MDCK gDNA <br>
** MDCK gDNA <br>
** HotDog Sample <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>
* NANO-C: Straw making with two plugs in progress, first lysis test with B.Subtillis cells planned for next week.<br>
*PATH: straw holder for Blackbird is machined  <br>
* Paper on Yeast: Extended Literature Review (12/11) <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>'''  
* New metal piece design set.<br>
* New metal piece - First week of Dec.<br>
* Evap paper 1st draft by the end of this week <br>
* Evap paper 1st draft by the end of this week <br>
<br>
<br>
Line 111: Line 94:
* Cathie submitted the companion BU IRB form for exemption. Revised, still still waiting. <br>
* Cathie submitted the companion BU IRB form for exemption. Revised, still still waiting. <br>
<br>
<br>
'''‡ PATH Grant (Lead:Cathie, Team:Frank, Sean, Mark, Jake Trueb (ME), Suma)''' <br>
'''‡ PATH Grant (Lead:Cathie, Team:Frank, Sean, Mark, Jake Trueb (ME))''' <br>
* Phone meeting with PATH this week. 10/13. <br>
* Phone meeting with PATH this week. 10/13. <br>
* Senior Project Proposals due in less than one week! (23 Nov.) <br>
* Senior Project Proposals submitted; proposal presentations Friday after Thanksgiving (Dec. 4).<br>
* Frank: Unfortunately MIA due to interviews.  14-day unstabilized storage PCR results to be analyzed very soon.<br>
* Frank: 7- and 14-day unstabilized RNA results emailed to group last week.
* Frank: PVA arrived. Experiments to commence shortly.<br>
* Frank: PVA experiments to begin after Thanksgiving.<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: 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 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>
* 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>
<br>
‡IIH Senior Project.
* Trouble locating PMMA sheet. <br>
* Try Dow Chemical Website. FOOD safe, FDA grade. <br>




Revision as of 13:20, 24 November 2009

Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

24 November 2009 Lab Meeting

  • Attending:
  • Missing:

‡ Announcements

  • Oakridge deadline is 1 Feb 2010.


‡ Flu R01:Integration


† HDA (Lead: Jaephil, Team:Sonali)

  • Start planning R01 for Submission on 2/5/10. MM, JD, CMK.
  • Dry reagent storage for RT and HDA - COP test well array provided fo Sonali
  • 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. Cathie will do Wed.


† 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. 3M GUSCN stands at pH 4.2 very acidic (some NaOH)
  • "Measure again 1:1 with DEPC water, 1:1 with PBS"
  • Silica nanoparticles from Fluka. 15nm "order"
  • Still working on the 150nm particles 1X experiments with lambda. HM


  • PLanning net silica per channel experiments. with 700nm, 3x possible. Deliver around 11/5/09.


  • 150nm and 700nm silica with 0.5mm (3.6uL SPE) channels.
  • 150nm Silica did not improve Ct on average, but one channel with 700nm silica performed exceptionally well, which brought the average up.
  • "relationship between PFU/ml and copy # of RNA. Do this via a serial dilution of Virus, put through Qiagen kit. Assuming that Qiagen is 100% efficient."
  • "Jessie get trained for real on SEM.*"

† PCR - CMI (Lead: Qingqing)

  • QQ will work on the initial integration steps of SPE + RT (reservoir)+ PCR. 
 - SPE+RT+PCR chip is designed.
  • PCR of C.Difficile DNA
 - toxin A.
   HDA assay for toxin A is tested in tube. It works
   HDA chip will be designed, and tested.
   SPE DNA from lisa for test?
 - toxin B.   
   on chip PCR works.
  • 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)
  • "ASB will give action items."


‡ Agilent Automated Sample Preparation (Lead: Alex)

  • So far tested on SNAP2:
    • B. subtilis gDNA
    • B. subtilis cells, high Cts for the range tested.
  • 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 next week.
  • Paper on Yeast: Extended Literature Review (12/11)
  • PATH: straw holder for Blackbird is machined

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

  • New metal piece - First week of Dec.
  • 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))

  • Phone meeting with PATH this week. 10/13.
  • Senior Project Proposals submitted; proposal presentations Friday after Thanksgiving (Dec. 4).
  • Frank: 7- and 14-day unstabilized RNA results emailed to group last week.
  • Frank: PVA experiments to begin after Thanksgiving.
  • 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?).


‡IIH Senior Project.

  • Trouble locating PMMA sheet.
  • Try Dow Chemical Website. FOOD safe, FDA grade.



Retrieved from "https://openwetware.org/mediawiki/index.php?title=Klapperich_Lab:Notebook/Lab_Meeting_Notes/2009/11/24&oldid=369698"