12 April 2011 Lab Meeting
- Short meeting today 10:30-12P
- Cathie at BU nominally T and W. At MIT M, Th and F.
- Microscope upgrade - will get package from Vermont Optechs this week
- Need help with a few MCF7 PCRs for colleague at NEU.
- People coming/people going, summer plans.
- Need protocol chapter from Andy and Sam.
†Trapping Mycobacteria (Jason)
- Running induction experiments; testing different engineered strains of mycobacteria.
- Within next few weeks hope to have good timecourse data for several different transcription factor targets.
†Nanomedicine (Sunil, Sonali, Cathie and Jen Rosen)
- mir 222 and RNU44 loci complete. Could not replicate Hyun's data down to 5 cells. We get down to 500 cells (3 channels)
- Down to 50 cells (2 channels). DNase treatment of RNA for mRNA RT-PCR will begin once DNAse inhibitor arrives. Allows bypass of (1) RNA precipitation and phenol/chloroform purification after DNAse treatment -- would likely lose much of the pg quantities.
- Allows bypass of heat inactivation DNASe treatment requiring high EDTA (5-15mM EDTA)which will inhibit RT-PCR.
- MgCl2 optimization done and would require maximum allowable of 14.75mM added to PCR mix for amplification. Cannot use Ct data for mRNA GAPDH then since the amplification curves were not reproducible for replicates. Results would be gel based (+/-).
†Wave 80 project (Andy/Sam/Sonali/Cathie)
- Every other week meeting 2-3:30 SKPYE.
- "Thawed" bDNA kit does not work, some reagent or condition was damaged while at room temperature for ~2 days. Still trying to diagnose the problem; probably not the label probe. (SB)
- Varied % ethanol washes and wash volumes for glycogen ppt in tube; no major difference in tube so can potentially use smaller wash volumes. Will repeat in monolith and compare results. Average recovery ~45%. (SB)
- Sent Wave 80 "dirty" samples with various wash volumes and new Pol synthetic RNA. (SB)
- Varying blood lysis time for glycogen ppt capture with "BoomD" lysis buffer. 15 minutes seems to be best. Will repeat with HIV particles.(SB)
- Will try out spiking super-concentrated (1e8 copies/mL) Seracare standards into blood (AF)
- Cloned whole-gag gene; making synthetic RNA for Wave80 (AF)
- Trying out new PCR primers to get lower -RT noise floor in Taqman assays
- Trying out exogenous (Xenopus Cdc6) synthetic RNA control (AF)
- Observed tip extraction procedure and following differences were noted between tube and straw/tip protocols (MM)
- initial tip wash with 100% ethanol at beginning of procedure. If done at least 1 day ahead then minor effect(better drying)
- SiO2 (pH2) not vortexed to mix with L6 lysis buffer (pH 6.4) -- affects pH and water content
- All mixes by tube inversions instead of vortexing. SiO2 pellets easily
- virus particles on ice > 30-1hr instead of quick thaw at 37C and immediate mix with GuSCN -- RNA degradation
- Viral lysis minimized. 10 min, not 15 min. 1 tube inversion in between, not inverted every 3 min (5 times)
- Multiple loading of sample in 300ul volumes until sample is completely through (minor)
- RNA in L6 lysis buffer 20 min - 1 hr depending on volume of SiO2 used in tip
- time to completion from virus contact with L6 was 1 hr - over 3hr -- RNA degradation once GuSCN is removed.
- Less washing -- half volume (450ul) used for all washes -- L2, 70% ethanol, Acetone -- residual GuSCN may be present
- No mixing of silica with wash reagents or water -- residual GuSCN may be present; elution efficiency
- Air drying -- no heat(56C) for drying off acetone before elution (minor)
- Elution with water at room temp without incubation vs. 10 min at 56C, then vortex -- decreased eluted RNA
- Elution volume much larger -- 80-100ul collected instead of 30ul collected. Unknown volume soaked through tip vs. 50ul in tube
-- Diluted RNA for downstream use
- Small changes may reduce some of these differences.
‡ Flu R01:Integration
- Paper drafting v.20?
- Grant renewal scored but not a fundable score. Revision submitted 3/5
† Sample Concentration (Lead: Jane, Nga)
- Meeting Fridays PHO 11-12pm.
- George gave Jane a few IRIS chips to fit into the evaporator. (A simple integration was done. Jane will take more pictures to send around)
- Jane and George try some concentrated VSV sample to try on real IRIS chips. (Jane did 2 samples and gave to George, results from IRIS and plaque assay pending)
- Jane did 4 runs with BSA coated chips and gave to John, plaque assay pending.
- John put Jane on John’s IBC for viral purification, pending review in March
- SimPore did demo presentation. They brought 2 Cyto Vu slides for each lab, they will send 4 SepCon separation inserts at 30 nm. (they do not have them in 50 nm), 6 Free-standing 30 nm Nanoporous membranes for distribution.
- Attempting to make PS plaque and emboss seems to work. Need to work on micromilling to make air control layer. Then bonding.
- To do: PEG precipitation as positive control. Plaque assay.
‡ SERS Franhofer Project (Jared, Chris, Jake, Cathie)
* Meetings Wed 9-10am at Fraunhofer.
* Meeting with Holger to discuss macro / micro concentrator sample transfer.
‡C. diff Project (Cathie, Sonali, Satish, Shichu, QQ, Lisa J., Andy)
- Meeting times TBA.
- Run Taqman (toxinB)real-time standard curves (40 cycles), those "false positives" (E1, E2, G1) are underdetermined. But gel results still show product band.
- Run Taqman (toxinB)real-time for 30 cycles instead of 40 cycles, those "false positives" (E1, E2, G1) are underdetermined, and no product band with gel.
- Run the HDA reactions for shorter times in tube 30 min, the "false positives" (E1, E2) don't show product band with gels
‡ CIMIT (actually two sepsis projects)- Sepsis (Cathie)
- NEW person needed for this project.
- Negative control of 16s primers as well as of DXS primers (Sonali's Mix) show signals on qPCR.
‡ PATH Grant (Lead:Cathie, Team:Jake, Samantha)
- Meetings Wed 3-4 pm every other.
- Doing glycogen ppt in monolith with blood varying wash volumes. (SB)
- Varying monolith volume to increase flow-rates and see effect on recovery with glycogen ppt. (SB)
- Do multiple small elutions from monolith to determine where/when RNA elutes. (SB)
- Starting storage studies in blood with Gag and Versant standard RNA this week!!! (SB)
- Prototype fabrication completed (mostly). Waiting on in line check valves (back ordered, will ship on 2/22) for testing. Current experiments are being conducted using manual stopcocks as temporary replacements. This works to some extent, but the larger internal volumes are causing fluid trapping issues. This issue will be mitigated somewhat with replacement with the correct check valves, but it may be necessary to redesign the syringe connection component in order to move the valves closer to the fluid union (thereby reducing potential trapped volume ).(JT)
- Prototype can handle ~2 mL total reagent volume. Larger reagent volumes are still possible, but may require a redesign of the waste collection unit.
- Extractions on Spiked Blood in prototype need to happen by mid March (JT)
- Small fluid bubbles show a tendency to become trapped at the pipette tip / o-ring interface. Adding bevels to the input port has lessened this phenomenon but not eliminated it. (JT)