8 February 2011 Lab Meeting
- QQ presenting her committee meeting talk practice on 2/22.
- Senior project teams presenting on 3/8, 10-15 min. each team.
- Cathie at BU nominally T and W. At MIT M, Th and F.
- Xin Brown wants us to throw away our PCR plates in her -20 freezer next to the RT machine (MM)
- Undergrads need more training for working in BL2 hoods. Bill (EHS) wants to know if we want 30-40min BL2 training for lab members. Anyone working with new students or training others, follow the BL2 hood SOP posted on the hood as per EHS instructions
- -80 racks arriving this week for 709. Move all DNA, bacterial stocks, bacteria sepsis to 720 -80. Make space in 720 -80 and update freezer maps on doors. Will announce which day this week. Stay tuned (MM)
†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.
- Started glycogen ppt for capture in tips: seems to work, no glycogen in flow-thru but variable capture results. (SB)
- Began glycogen ppt in tips with Gag RNA spiked into human whole blood: seems to work, no glycogen in flow-thru. (SB)
- Prepared new silica for sending Wave 80 "dirty" samples with various wash volumes; will send before next Wave 80 meeting. (SB)
- Compare glycogen ppt with and without PVA stablizer over short time periods. (SB)
- Varying blood lysis time for glycogen ppt capture with "BoomD" lysis buffer. (SB)
- Re-doing blood lysis with new SiO2 prep (AF)
- Will try out spiking super-concentrated (1e8 copies/mL) Seracare standards into blood (AF)
- Cloning for whole-gag gene for synthetic RNA production for Wave80 (AF)
- Trying to figure out discrepancies between my post blood-lysis RNA recovery (in tube) vs. Sam's (AF)
- 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.8
- Grant renewal scored but not a fundable score. Revision for July 5.
† HDA (Lead:Sonali)
† 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.
‡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.
- Glycogen ppt in tips with and without blood seems to work: no glycogen in flow-thrus but variable recovery results. (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)