Klapperich Lab:Notebook/Lab Meeting Notes/2010/03/04: Difference between revisions
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=4 March 2010 Lab Meeting= | |||
Alex Presentation. | |||
'''‡ Announcements'''<br> | |||
'''‡ Flu R01:Integration''' <br> | |||
* meetings every other M 1-3pm. 705. <br> | |||
† HDA (Lead: Jaephil, Team:Sonali)<br> | |||
* Patient Samples - dilution experiments (MM/SH/JD) <br> | |||
* Start planning R01 for Submission on '''6/5/10.''' MM, JD, CMK. <br> | |||
* HDA mix in dried form - Expecting it from BH. <br> | |||
* warm start on the way <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> | |||
<br> | |||
† Sample Concentration (Lead: Jane, Team: Jaephil) <br> | |||
* Air resistance in the air channel is too large. <br> | |||
** New branched "membrane" chip design #1 in lithography stage. <br> | |||
** New "tube" chip design in drawing stage. Obtained more porous PTFE tubing samples <br> | |||
* Samples expected from Fauchet group. <br> | |||
<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> | |||
<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 <br> | |||
** talk about PCR improvements BEFORE we use new patient samples. <br> | |||
* PCR1 will combine with PCR2 if PCR2 comes back for revisions. (CMK)<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) | |||
<br> | |||
† 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. MEETINGS CANCELLED THIS 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) -- DONE 2/25 br> | |||
<br> | |||
'''‡ Agilent Automated Sample Preparation (Lead: Alex)'''<br> | |||
* Paper on HotDog - with CMK/ASB <br> | |||
<br> | |||
'''‡ COBRA (Lead: Jaephil, Team: Cathie, Jane for virus only, PHO folks)<br>''' | |||
* Evap paper - Additional experiment for Fig 3. By 3/15. <br> | |||
<br> | |||
'''‡ Biointerfaces group (Lead: MinCheol, Team: Cathie, MCK, Wong and Meller folks)'''<br> | |||
'''* Cathie will submit paper.<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: One zero-time 6-straw set run with 5-25% efficiency. Second set in progress (24 Feb).<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). Testing has been very slow-going due to an excess amount of other school work/tests. <br> | |||
* 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.<br> | |||
<br> | |||
'''‡ IIH Senior Project.'''<br> | |||
* Progress Report 1 completed | |||
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4 March 2010 Lab MeetingAlex Presentation. ‡ Announcements ‡ Flu R01:Integration
† HDA (Lead: Jaephil, Team:Sonali)
* wrong size products(55bp). * no designed size product.
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