Klapperich Lab:Notebook/Lab Meeting Notes/2009/11/17

17 November 2009 Lab Meeting

• Attending:
  
• Missing:
  

‡ Announcements

• Oakridge deadline is 1 Feb 2010.
  
• PAPERS Drafted before MicroTAS Are we done with this? 1st draft by Thur
  

Evap paper 1st draft by the end of this week

‡ Flu R01:Integration

• New design - address evaporation loss, Reagent delivery and storage solution. Consider marginating flows?
  
• New design sent to make Rubber mold: outline octagon problem. Revised design sent yesterday.
  

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

• MDCK Cells are not propagating. Try troubleshooting efforts. Sonali will do on Monday.
  
  • Cells are fine. Problem was no water in incubator pan. Trained Hussam and Jessie on how to make more MDCK liquid N2
    
  • stock. Now have 25 more MDCK tubes of 5X10^6 cells in 1 mL aliquots frozen on 11/4/09
    
• Cassidy needs to see plaque assay again - so when cells are up, she needs training.
  
  • Cassidy trained by Brendan. Will do 1 more plaque assay with Jane to complete training.
    
• Jane will train on Wednesday and Thursday this week.
  
• Up to high 10^7 for positive control. Silver substrate no signal.
  
  • 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
  
• Main loss is at the outlet/sample collection: tangential filtration design in drawing stage. - no tangential flow. New design incorporates Lee valve, no sample collection and dispensing necessary.
  
• Set up COMSOL and StarCD, look for governing equations for simulation of evaporation. - ongoing
  
• Committee meeting setup
  

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

• 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 DNA was used.
 -it works!
 -patient sample will be tested next.
 

• PCR of C.Difficile DNA

 - primer for toxin A.
    - 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 14.
   -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.
  

† 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. New mold design sent.
  
• Paper submitted 10/6.
  

‡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)

• Metal piece modification for the integration - Fix did not turned out good. Make new or try fix again?
  
• paper 1, 1st draft by Thu(10.29): Evap with Sol Gel substrate. Not integrated. MSSA, E coli.
  

‡ 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?).