16 August 2010 Lab Meeting
""* EVERYONE bring summary slides (1-3 only).
- Lab meeting same time fall M 1-3pm
†Trapping Mycobacteria (Jason)
- Can trap and induce cells every time, and watch them turn off after switching to non-inducing media.
- Having trouble achieving a second induction (On/Off/On). Concerns about 'health' of cells after 5-6 hours in device.
- Cells at 37C, but I don't see cell division even though experiment lasts for 2 doubling times.
- Ordered a standard Invitrogen live-dead assay kit to help figure out what's going on.
- --> Has anyone in the room tried doing the live/dead on-chip before? I don't think it will be too difficult to do.
Old summary slides for Jason (from 8/2): Image:JasonSlidesModified.ppt
†Wave 80 project (Andy/Jane)
- Did 1 baseline run in parallel for intact particles, RNA from SPE, and RNA from Qiagen.
- Will start making plasmid templates for extraction process monitoring using real-time PCR (backup to using only bDNA assay strips)
- SPE on Std B repeated. Chip problem. Plan to make more chips and repeat SPE on Std B and C.
‡ Flu R01:Integration
* Weekly meetings Tuesday 3-4 pm.
- List people required to attend meeting: MM, JC, Brendan, Ahjegannie, Chris? Send notes to QQ, MM will brief her when she's back
- 1) 73 (+) and 73 (-) samples(Cathie)
- 2) Key pieces of data needed for each sample:PFU/ml for a subset of samples, tube PCR & copy #/ml all, chip PCR all, TC all
- 3) Jessie will input the inventory (# samples from BUMC, #samples from BIDMC, break down by the number of aliquots per sample)
- 4) Use BIDMC samples based on ones Nira P. has culture/PCR data for.
- 5) JC -- PFU/ml, copy number PCR; MM -- culture and RNA extractions for JC copy# PCR; BC, QQ, AS, CH -- chip PCR
- 6) Set timelines for each task for each person
- for PCR just do more reactions, pool and etoh precipitate.
- For HDA, re-try with min-elute PCR purification kit to concentrate product, remove dimers for TOPO TA (MM).
- Warm start enzyme -- less dimers but waters always come up positive for E.coli. Go back to regularly sold enzyme IsoAmp II. Get dimers, but difference in size (50-70 bp) allows for distinguishing from products (113bp)on gels.
- Warm start enzyme is causing false positives -- New primers, New water, new kit, RNA hood in BL2 lab used with RNA pipets
- Use on bleached cut chips, with 2X enzyme amount and 0.05% BSA blocking on dry bath
- Dry heat bath, thermal conductive tape, insulator and styrofoam box on chip to set temp at 65C +/- 0.5 after 20 min.
- Check other methods of heating (SH).
† HDA (Lead:Sonali)
- Start planning R01 for Submission on 10/5/10. MM, CMK.
- HDA mix in dried form - Expecting it from BH -- ON HOLD.
- QQ preliminary chip work
- Sequence all output -- see cloning section above
- 2nd HDA design - "microSPE" + "Flu primer" + "HDA in a cup" + "Detection kit from Biohelix"
† Sample Concentration (Lead: Jane)
- Patient sample run in preparation: making new chips to solve nanoport problem
- VTM contains gelatin. Not suitable for evap. New patient sample in PBS obtained. Will make new chips to test.
† SPE Column Optimization for DNA/RNA. (Lead: Jessie)
- Patient samples: <10^4 cp/mL, ~10^6 cp/mL, and >10^5 cp/mL detection limit with SPE.
- RNA ladder: elute RNA length of 200 to 1500 bases, but not beyond (up to 6000 bases).
- Chitosan modified silica yielded <10% while regular channel yielded ~50% when purified flu RNA (from Qiagen extraction) is loaded.
† Integrated chip for flu(SPE+RT+PCR) (Lead: Qingqing)
- Real copy number of previous human samples. So QQ is working with the higher concentration samples. Range is 10^6-10^8 of copy number/ml.
- REVISIT the % recovery number.
- QQ tried: more enzyme, Tween 20, BSA amounts.
- integrated flu chip paper has been send to cathie, wait for revision.
- PCR1 will combine with PCR2 (CMK)
‡C. diff Project (Cathie, Sonali, Satish Singh, Lisa J., His post doc )
- Chip HDA reaction: Cloning successful with Patient #30 - derived HDA chip reactions w/ pGEM vector
- send SH the HDA in a cup paper draft from JD.(CMK)
‡ Coulter Flu Fraunhofer Project (Lead: Sonali, Team: Sonali, Jessie, Cathie, CMI Folks, Qingqing)
- Meetings Fridays/every other
‡ Automated Sample Preparation (Lead: Alex)
- Paper on HotDog - with CMK/ASB. 7/2
‡ COBRA (Lead: Jaephil, Team: Cathie, Jane for virus only, PHO folks)
- Evap paper - accepted!
- Cathie corrections today 8/2.
* Latest draft with Cathie.
‡ CIMIT- Sepsis (Lead:Cathie, Team: Alex)
- Had meeting with Prof. Hauser (7/9) and worked with Kiyo and Tolga in Harvard Lab on extracting DNA from Plasma with Qiagen kit.
- Preparing experimental plan for next steps.
- Got introduction to procedures for 720 and PCR room by Sonali.
- Things to clarify: -safety issues and regulations related to transport and work on human samples.
‡ PATH Grant (Lead:Cathie, Team:Jake, Samantha)
- new flow rate data for altered polymer formulation
- RNA extraction using altered polymer formulation - SPE 0 vs SPE 100 (it works!!!)
- upcoming experiments