11 February 2010 Lab Meeting
‡ Flu R01:Integration
- meetings every other M 1-3pm. 705.
† HDA (Lead: Jaephil, Team:Sonali)
- Start planning R01 for Submission on 6/5/10. MM, JD, CMK.
- HDA mix in dried form - Cathie found it
- LOD tests HDA in a cup - barely amplified at 3pg/mL
- HDA negative water reactions all have primer dimers/non-specific band at 70bp for c diff HDA (MM)
- 2nd HDA design - "microSPE" + "Flu primer" + "HDA in a cup" + "Detection kit from Biohelix"
† Sample Concentration (Lead: Jane, Team: Jaephil)
- New design transfered to COP
- Air resistance in the air channel is too large:
- Air pressure: 50 psi, Flow rate: 53 uL/hr, in room temperature. ie. evaporated 633 uL in 12hr. This is very slow
- Redesign channel to reduce air resistance: see video
- Required samples of porous Teflon tubing, need advice on tube/chip format
† SPE Column Optimization for DNA/RNA. (Lead: Sonali, Team: Hussam, Jessie, Brendan)
- Sonali put out a new RNA protocol (correct SPE batter, homemade buffers, less ethanol washing, RNA Secure) before break.
- Protocol tested with 10ng, 1ng, 0.1ng Stratagene purified human RNA (MM) and 100 uL of 1:64 dilution ATCC MDCK sup Flu A/PR/8/34 (JC)
- Human Total RNA see % yields at 50% and fraction 1 + fraction 2 = 4-5ng RNA out of 10ng with all changes. With high vol *ethanol wash but other protocol improvements, see 40% yield at all other concentrations.
- For Flu virus see improved Ct value by 7 cycles in fraction 1 from multiple channels
- Also detecting 1.4 logs more of virus at 1:64 JC virus prep than before.
- Switching from lambda phage DNA to TaqMan RNase P detection [update]
- Hussam Runs chip with RNA (confirm procedure is correct) [update]
- Jessie will run chips with serial virus dilutions from 1:1 to 1:1,000,000 to re-do previous dilution experimnent with WHO SYBR green assay on chip.
- Jessie will test 2mm wide channels with 4uL SPE with Black Beauty for efficiency.
- Sonali will assay BIDMC virus sample with newest protocol with 0.7uM Silica and compare with Qiagen RNA extraction with CDC PCR assay
† integrated chip for flu(SPE+RT+PCR) (Lead: Qingqing)
- integrated chip works with ATCC flu virus
- working on improving the efficiency
- testing real patient sample
- PCR1 Paper back to MCK this week.
- CMK: PCR 1 draft. Analytical Chem.
‡C. diff Project (Cathie, Sonali, Satish Singh, Lisa J., His post doc )
† PCR of C.Difficile DNA (Qingqing)
- ATCC C.dif DNA with Negative patient sample works on chip
- Another positive patient sample was tested on chip
* wrong size products(55bp).
* no designed size product.
‡ Coulter Flu Fraunhofer Project (Lead: Sonali, Team: Sonali, Jessie, Cathie, CMI Folks, Qingqing)
- New meeting time, T, 9:30 am every other week.
- Will be done by next Tuesday - PCR with 3 different kits of same serial dilution RNA sample (Invitrogen - SuperscriptIII Platinum, Ambion - AgPath-ID, Qiagen - OneStep RT-PCR Kit)
‡ Agilent Automated Sample Preparation (Lead: Alex)
- Paper on HotDog in progress.
‡ COBRA (Lead: Jaephil, Team: Cathie, Jane for virus only, PHO folks)
- Evap paper - Additional experiment for Fig 3.
‡ Biointerfaces group (Lead: MinCheol, Team: Cathie, MCK, Wong and Meller folks)
* Cathie will submit paper inquiry.
‡ CIMIT- Sepsis (Lead:Cathie, Team:TBA)
- Sample collection ongoing.
‡ PATH Grant (Lead:Cathie, Team:Frank, Sean, Mark, Jake Trueb (ME))
- Submitted Gantt Document to PATH 11/25.
- Frank: Repeating 1-week stabilized extractions today (4 Feb).
- Sean: Started making straws for the new experiments we talked about (DNA weights of 1ng, 10ng, and 100ng through 700nm at SPE 100 monolith at 0-day time point). New straw pieces are finished, but they may need some modification before they will fit in the Blackbird fixture.
- Mark: as of 4 Feb. has loaded 6 and 4 month trials with 10ng/straw. Results of the 1ng/straw 0-day trial inconclusive, redo possible. Sticking with 10ng/straw for remaining tests. Loaded 2 month trial on Tuesday. Will load shorter time periods next week (i.e. 3-day, 1 week). Straw fabrication ongoing.
‡ IIH Senior Project.
- ELISA kits have been delivered
- Protocol written for using cutter plotter. Will post to share drive
- Made "test" chips from 100 micron COP but cuts are sloppy
- Will change settings on cutter to clean up cuts
- First thought is to slow down speed
- Started acquiring images of the 100 micron "test" chips