28 January 2010 Lab Meeting
Jessie Presentation.
‡ Announcements
- Oakridge deadline is 1 Feb 2010. Have one from QQ- Anyone else?
- Shichu Huang will be joining us in Feb.
‡ 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.
- Jaephil trying out the lyophilizer with just water to save on HDA reagents. Then Sonali lyophilize HDA mixes +/- trehalose.(MM)
- HDA at 65, 66, 67, 68, 69, 70C done with C diff ATCC DNA. Works at all except 70C. Less amplification as Temp increases (MM)
- HDA negative water reactions all have primer dimers/non-specific band at 70bp for c diff HDA (MM)
- Jaephil to make COP chips for LOD expt with C diff DNA. (MM)
- 2nd HDA design - include RT, dry reagent, reduced evap loss, and parallel filling, etc
† Sample Concentration (Lead: Jane, Team: Jaephil)
- New design sent to make Rubber mold: outline octagon problem. Revised design sent yesterday.
- 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 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.
- Sonali will assay BIDMC virus sample with newest protocol with 0.7uM Silica and compare with Qiagen RNA extraction with CDC PCR assay
† PCR - CMI (Lead: Qingqing)
- 6th Integrated chip with SPE+RT+PCR chip was designed.
- "crack" issue is solved.
- It works for ATCC flu virus, however low efficiency.
- working on improving the efficiency
† PCR of C.Difficile DNA (Qingqing)
- Meeting today at 3:30pm
- Toxin A.
- HDA dilution experiments for toxin A has been tested in tube. The products has been tested in Bio-analyzer. 1pg is the limitation for the template, in order to get a detectable result.
- Toxin B.
- PCR assay works on chip with ATCC C.dif DNA.
- old SPE DNA has ct values above 30, on chip PCR with these DNA does not work either on 30 cycle chip nor on 40 cycle chip.
- Qiagen extracted DNA from Lisa on Jan 6 have ct values between 20-30.
- on chip PCR with lisa's DNA also does not work either on 30 cycle chip nor on 40 cycle chip.
- 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 )
‡ Coulter Flu Fraunhofer Project (Lead: Sonali, Team: Sonali, Jessie, Cathie, CMI Folks, Qingqing)
- New meeting time, T, 9:30 am every other week.
‡ Agilent Automated Sample Preparation (Lead: Alex)
- Paper on HotDog in progress.
‡ COBRA (Lead: Jaephil, Team: Cathie, Jane for virus only, PHO folks)
- New metal piece - First week of Dec.
- Evap paper -CMK edited, back to JD and JZ this week
‡ Biointerfaces group (Lead: MinCheol, Team: Cathie, MCK, Wong and Meller folks)
* Cathie will submit paper inquiry.
‡ CIMIT- Sepsis (Lead:Cathie, Team:TBA)
‡ PATH Grant (Lead:Cathie, Team:Frank, Sean, Mark, Jake Trueb (ME))
- Submitted Gantt Document to PATH 11/25.
- Frank: 1-week in-tube stabilization experiment successful. Extractions completed for 1-week storage. PCR of eluted samples in progress (28 Jan).
- Sean: Ready to start preparing the monoliths today (1.21) after the meeting. Will be able to make it to the meeting by 3 every week. Hussam will be training me on PCR next week. Need bench training still
- Mark: as of 26 Jan. has loaded 6 and 4 month trials with 10ng/straw. Pending results of the 1ng/straw 0-day trial will load 2 month trial next week and shorter time periods in weeks to come. Straw fabrication ongoing.
‡ IIH Senior Project.
- Ordering ELISA Kits
- Determining materials to test fluorescence
- Transparancy, COP, PDMS (gold-standard)
- material list from study (Xurography, Daniel A. Bartholomeusz, p 1367)
- Planned to reconnect with Jose, he is out of town until feb.
- Returning to the cutter plotter at MIT for 'origami testing'
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