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=25 February 2010 Lab Meeting= | |||
Jaephil Presentation. | |||
'''‡ Announcements'''<br> | |||
* Oakridge deadline is Confusing. The website says something different from the email. Have one from QQ- Anyone else? <br> | |||
* DNA Isolation Assay Review. [[Media:Please see attached file.]] [[Image:[[Example.jpg]]]]<br> | |||
<br> | |||
'''‡ Flu R01:Integration''' <br> | |||
* meetings every other M 1-3pm. 705. <br> | |||
† HDA (Lead: Jaephil, Team:Sonali)<br> | |||
* Start planning R01 for Submission on '''6/5/10.''' MM, JD, CMK. <br> | |||
* HDA mix in dried form - Cathie found it<br> | |||
* HDA in tube works up to 69C. Negative at 70C. 65C is best. No temps below 65C tested. Confirms manufacturer papers and protocol (MM) <br> | |||
* 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 <br> | |||
* HDA negative water reactions all have primer dimers/non-specific band at 70bp for c diff HDA (MM)<br> | |||
* 2nd HDA design - "microSPE" + "Flu primer" + "HDA in a cup" + "Detection kit from Biohelix"<br> | |||
† Sample Concentration (Lead: Jane, Team: Jaephil) <br> | |||
* New design transfered to COP <br> | |||
* Air resistance in the air channel is too large: <br> | |||
** Air pressure: 50 psi, Flow rate: 53 uL/hr, in room temperature. ie. evaporated 633 uL in 12hr. This is very slow <br> | |||
* Redesign channel to reduce air resistance: see video <br> | |||
** Required samples of porous Teflon tubing, need advice on tube/chip format <br> | |||
† SPE Column Optimization for DNA/RNA. (Lead: Sonali, Team: Hussam, Jessie, Brendan) <br> | |||
* Sonali put out a new RNA protocol (correct SPE batter, homemade buffers, less ethanol washing, RNA Secure) before break. <br> | |||
* 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) <br> | |||
* 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. <br> | |||
*For Flu virus see improved Ct value by 7 cycles in fraction 1 from multiple channels <br> | |||
*Also detecting 1.4 logs more of virus at 1:64 JC virus prep than before. <br> | |||
*Switching from lambda phage DNA to TaqMan RNase P detection [update] <br> | |||
* Hussam Runs chip with RNA (confirm procedure is correct) [update]<br> | |||
* 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.<br> | |||
* 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. <br> | |||
* 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 <br> | |||
* 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. <br> | |||
† integrated chip for flu(SPE+RT+PCR) (Lead: Qingqing) <br> | |||
* integrated chip works with ATCC flu virus <br> | |||
* working on improving the efficiency | |||
* testing real patient sample | |||
* PCR1 Paper back to MCK this week. <br> | |||
* CMK: PCR 1 draft. Analytical Chem. <br> | |||
<br> | |||
'''‡C. diff Project''' (Cathie, Sonali, Satish Singh, Lisa J., His post doc )<br> | |||
* See email notes. <br> | |||
* See HDA notes above for styrofoam cup/chip work (MM) | |||
† PCR of C.Difficile DNA (Qingqing)<br> | |||
* 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. | |||
<br> | |||
'''‡ Coulter Flu Fraunhofer Project (Lead: Sonali, Team: Sonali, Jessie, Cathie, CMI Folks, Qingqing)''' | |||
* New meeting time, T, 9:30 am every other week. <br> | |||
* 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)<br> | |||
<br> | |||
'''‡ Agilent Automated Sample Preparation (Lead: Alex)'''<br> | |||
* Paper on HotDog (Draft) - still working on it. But almost done. <br> | |||
'''‡ COBRA (Lead: Jaephil, Team: Cathie, Jane for virus only, PHO folks)<br>''' | |||
* Evap paper - Additional experiment for Fig 3. <br> | |||
<br> | |||
'''‡ Biointerfaces group (Lead: MinCheol, Team: Cathie, MCK, Wong and Meller folks)'''<br> | |||
'''* Cathie will submit paper inquiry.<br>''' | |||
<br> | |||
'''‡ CIMIT- Sepsis (Lead:Cathie, Team:TBA)'''<br> | |||
* Sample collection ongoing. <br> | |||
<br> | |||
'''‡ PATH Grant (Lead:Cathie, Team:Frank, Sean, Mark, Jake Trueb (ME))''' <br> | |||
* Submitted Gantt Document to PATH 11/25. <br> | |||
* Frank: Eluting/PCR on extra-drying one-week samples. Also extracting/eluting/PCRing appropriate controls (stored in-solution, then straw extraction before PCR).<br> | |||
* 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. <br> | |||
* Mark: as of 18 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. Straw fabrication ongoing.<br> | |||
<br> | |||
'''‡ IIH Senior Project.'''<br> | |||
* Progress Report 1 completed | |||
* Began making 100 micron chips with revised design | |||
** Initial imaging shows cleaner cuts but still difficult to distinguish layers | |||
Revision as of 14:12, 24 February 2010
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25 February 2010 Lab MeetingJaephil Presentation. ‡ Announcements
† HDA (Lead: Jaephil, Team:Sonali)
† Sample Concentration (Lead: Jane, Team: Jaephil)
† SPE Column Optimization for DNA/RNA. (Lead: Sonali, Team: Hussam, Jessie, Brendan)
† integrated chip for flu(SPE+RT+PCR) (Lead: Qingqing)
† PCR of C.Difficile DNA (Qingqing)
* wrong size products(55bp). * no designed size product.
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