25 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 - Expecting it from BH.
- HDA in tube works up to 69C. Negative at 70C. 65C is best. No temps below 65C tested. Confirms manufacturer papers and protocol (MM)
- LOD tests completed by MM on chip/in cup for 10ng, 0.3ng, 0.03ng, 0.003ng, 0.0003ng. Amplicon present down to 3pg, though very faint. Negative at 0.3pg
- 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)
- Air resistance in the air channel is too large.
- New "branched" membrane chip design #1 in lithography stage.
- New "tube" chip design in drawing stage. Obtained more porous PTFE tubing samples
† 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 tried 4 2mm channels with 4uL SPE on Black Beauty. Only one sucessful elution with 70uL in two fractions. Got 1E6 copies compared to 8E6 previously.
- Sonali completed 5 total patients (BUMC and BIDMC) extracted and RT-PCR'd successfully using new RNA protocol and small SPE channel chips. All samples selected based on Qiagen kit extractions having low Cts at 15-20 cycles
- Sonali gave Qingqing 3 of these patient RNAs extracted on chip SPE for testing on-chip RT-PCR with SuperScript vs Qiagen one-Step kit.
† 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 will combine with PCR2 if PCR2 comes back for revisions. (CMK)
‡C. diff Project (Cathie, Sonali, Satish Singh, Lisa J., His post doc )
- See email notes.
- See HDA notes above for styrofoam cup/chip work (MM)
† 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. MEETINGS CANCELLED THIS 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 (Draft) - still working on it. But almost done.
‡ 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: One zero-time 6-straw set run with 5-25% efficiency. Second set in progress (24 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). Testing has been very slow-going due to an excess amount of other school work/tests.
- Mark: as of 25 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 2/2. 1-week trial eluted Monday 2/15 again inconclusive - most likely there was a problem with the straws, a bad lot (same lot as the 1ng/straw test). To redo 1-week trial in the future with new lot of straws. Also troubleshooting with smaller wash volumes (down to 50uL) with results on 2/25. Straw fabrication ongoing.
‡ IIH Senior Project.
- Progress Report 1 completed
- Began making 100 micron chips with revised design
- Initial imaging shows cleaner cuts but still difficult to distinguish layers