16 August 2010 Lab Meeting

‡ Announcements

• No presentation.
  

""* EVERYONE bring summary slides (1-3 only).

• Lab meeting same time fall M 1-3pm
  

†Trapping Mycobacteria (Jason)

• Can trap and induce cells every time, and watch them turn off after switching to non-inducing media.
• Having trouble achieving a second induction (On/Off/On). Concerns about 'health' of cells after 5-6 hours in device.
• Cells at 37C, but I don't see cell division even though experiment lasts for 2 doubling times.
• Ordered a standard Invitrogen live-dead assay kit to help figure out what's going on.
• --> Has anyone in the room tried doing the live/dead on-chip before? I don't think it will be too difficult to do.

Old summary slides for Jason (from 8/2): Image:JasonSlidesModified.ppt

†Wave 80 project (Andy/Jane)

• Did 1 baseline run in parallel for intact particles, RNA from SPE, and RNA from Qiagen.
• Will start making plasmid templates for extraction process monitoring using real-time PCR (backup to using only bDNA assay strips)
• SPE on Std B repeated. Chip problem. Plan to make more chips and repeat SPE on Std B and C.

‡ Flu R01:Integration
* Weekly meetings Tuesday 3-4 pm.

• List people required to attend meeting: MM, JC, Brendan, Ahjegannie, Chris? Send notes to QQ, MM will brief her when she's back
  
• 1) 73 (+) and 73 (-) samples(Cathie)
  
• 2) Key pieces of data needed for each sample:PFU/ml for a subset of samples, tube PCR & copy #/ml all, chip PCR all, TC all
  
• 3) Jessie will input the inventory (# samples from BUMC, #samples from BIDMC, break down by the number of aliquots per sample)
  
• 4) Use BIDMC samples based on ones Nira P. has culture/PCR data for.
• 5) JC -- PFU/ml, copy number PCR; MM -- culture and RNA extractions for JC copy# PCR; BC, QQ, AS, CH -- chip PCR
• 6) Set timelines for each task for each person

• • SEQUENCING WORK
    
• for PCR just do more reactions, pool and etoh precipitate.
  
  • For HDA, re-try with min-elute PCR purification kit to concentrate product, remove dimers for TOPO TA (MM).
    
  • Warm start enzyme -- less dimers but waters always come up positive for E.coli. Go back to regularly sold enzyme IsoAmp II. Get dimers, but difference in size (50-70 bp) allows for distinguishing from products (113bp)on gels.
    
  • Warm start enzyme is causing false positives -- New primers, New water, new kit, RNA hood in BL2 lab used with RNA pipets
    
  • Use on bleached cut chips, with 2X enzyme amount and 0.05% BSA blocking on dry bath
    
  • Dry heat bath, thermal conductive tape, insulator and styrofoam box on chip to set temp at 65C +/- 0.5 after 20 min.
    
• Check other methods of heating (SH).
  

† HDA (Lead:Sonali)

• Start planning R01 for Submission on 10/5/10. MM, CMK.
  
• HDA mix in dried form - Expecting it from BH -- ON HOLD.
  
• 
  
• QQ preliminary chip work
  
• Sequence all output -- see cloning section above
  
• 2nd HDA design - "microSPE" + "Flu primer" + "HDA in a cup" + "Detection kit from Biohelix"
  

† Sample Concentration (Lead: Jane)

• Patient sample run in preparation: making new chips to solve nanoport problem
  
• VTM contains gelatin. Not suitable for evap. New patient sample in PBS obtained. Will make new chips to test.
  

† SPE Column Optimization for DNA/RNA. (Lead: Jessie)

• Patient samples: <10^4 cp/mL, ~10^6 cp/mL, and >10^5 cp/mL detection limit with SPE.
• RNA ladder: elute RNA length of 200 to 1500 bases, but not beyond (up to 6000 bases).
• Chitosan modified silica yielded <10% while regular channel yielded ~50% when purified flu RNA (from Qiagen extraction) is loaded.

† Integrated chip for flu(SPE+RT+PCR) (Lead: Qingqing)

• Real copy number of previous human samples. So QQ is working with the higher concentration samples. Range is 10^6-10^8 of copy number/ml.
  
• REVISIT the % recovery number.
  
• QQ tried: more enzyme, Tween 20, BSA amounts.
  
• integrated flu chip paper has been send to cathie, wait for revision.
  
• PCR1 will combine with PCR2 (CMK)
  

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

• Chip HDA reaction: Cloning successful with Patient #30 - derived HDA chip reactions w/ pGEM vector
• send SH the HDA in a cup paper draft from JD.(CMK)
  

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

• Meetings Fridays/every other
  

‡ Automated Sample Preparation (Lead: Alex)

• Paper on HotDog - with CMK/ASB. 7/2

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

• Evap paper - accepted!
  
• Cathie corrections today 8/2.
  

‡ Biointerfaces
* Latest draft with Cathie.

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

• Had meeting with Prof. Hauser (7/9) and worked with Kiyo and Tolga in Harvard Lab on extracting DNA from Plasma with Qiagen kit.
  
• Preparing experimental plan for next steps.
  
• Got introduction to procedures for 720 and PCR room by Sonali.
  
• Things to clarify: -safety issues and regulations related to transport and work on human samples.
  

‡ PATH Grant (Lead:Cathie, Team:Jake, Samantha)

• new flow rate data for altered polymer formulation
  
• RNA extraction using altered polymer formulation - SPE 0 vs SPE 100 (it works!!!)
  
• upcoming experiments