Klapperich Lab:Notebook/Lab Meeting Notes/2010/02/11: Difference between revisions

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* Start planning R01 for Submission on '''6/5/10.''' MM, JD, CMK. <br>
* Start planning R01 for Submission on '''6/5/10.''' MM, JD, CMK. <br>
* HDA mix in dried form - Cathie found it<br>
* HDA mix in dried form - Cathie found it<br>
* LOD tests HDA in a cup - barely amplified at 3pg/mL<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>
* 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>  
* 2nd HDA design - "microSPE" + "Flu primer" + "HDA in a cup" + "Detection kit from Biohelix"<br>  
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* New design transfered to COP <br>
* New design transfered to COP <br>
* Air resistance in the air channel is too large: <br>
* Air resistance in the air channel is too large: <br>
** Air pressure: 50 psi, Flow rate: 53 uL/hr, in room temperature. This is very slow <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>
* Redesign channel to reduce air resistance: see video <br>
** Required samples of porous Teflon tubing, need advice on tube/chip format <br>
** Required samples of porous Teflon tubing, need advice on tube/chip format <br>
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* Hussam Runs chip with RNA (confirm procedure is correct) [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 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 will test 2mm wide channels with 4uL SPE with Black Beauty for efficiency. <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 will assay BIDMC virus sample with newest protocol with 0.7uM Silica and compare with Qiagen RNA extraction with CDC PCR assay <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 for flu(SPE+RT+PCR) (Lead: Qingqing) <br>
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<br>
<br>
'''‡C. diff Project''' (Cathie, Sonali, Satish Singh, Lisa J., His post doc )<br>
'''‡C. diff Project''' (Cathie, Sonali, Satish Singh, Lisa J., His post doc )<br>
* See email notes.  
* See email notes. <br>
* See HDA notes above for styrofoam cup/chip work (MM)
† PCR of C.Difficile DNA (Qingqing)<br>
† PCR of C.Difficile DNA (Qingqing)<br>
* ATCC C.dif DNA with Negative patient sample works on chip
* ATCC C.dif DNA with Negative patient sample works on chip
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<br>
<br>
'''‡ Agilent Automated Sample Preparation (Lead: Alex)'''<br>
'''‡ Agilent Automated Sample Preparation (Lead: Alex)'''<br>
* Paper on HotDog in progress.  <br>
* Paper on HotDog (Draft) - still working on it. But almost done.  <br>




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'''‡ PATH Grant (Lead:Cathie, Team:Frank, Sean, Mark, Jake Trueb (ME))''' <br>
'''‡ PATH Grant (Lead:Cathie, Team:Frank, Sean, Mark, Jake Trueb (ME))''' <br>
* Submitted Gantt Document to PATH 11/25. <br>
* Submitted Gantt Document to PATH 11/25. <br>
* Frank: Repeating 1-week stabilized extractions today (4 Feb).<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>
* 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 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.<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>
<br>
'''‡ IIH Senior Project.'''<br>
'''‡ IIH Senior Project.'''<br>
* ELISA kits have been delivered
* Progress Report 1 completed
* Protocol written for using cutter plotter. Will post to share drive
* Began making 100 micron chips with revised design
* Made "test" chips from 100 micron COP but cuts are sloppy
** Initial imaging shows cleaner cuts but still difficult to distinguish layers
** 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
 


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Revision as of 07:20, 18 February 2010

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11 February 2010 Lab Meeting

Jaephil Presentation.

‡ Announcements


‡ 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
  • 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)

  • 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 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 Paper back to MCK this week.
  • CMK: PCR 1 draft. Analytical Chem.


‡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.
  • 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: Eluting/PCR on extra-drying one-week samples. Also extracting/eluting/PCRing appropriate controls (stored in-solution, then straw extraction before PCR).
  • 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 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.


‡ 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


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