9:50 am – 7:00 pm

Project 2

• see post

10:00 am – 9:00 pm

To do

• check to see if there are any notifications from Judy about K99
• check if XZ’s Biosketch was sent
• book hotel for Fly meeting!
• abstract for CSHL genome biology meeting
• READ Ph paper draft and send comments to team
• project 2 data analysis as per yesterday’s discussion

Ph Project

Cell counts

• Ph in S2, 06-12-2013 data: 42 cells
• PhF in PhWT cells, 2013-06-12 data: 22 cells
• PhF in PhML cells, 2013-06-12 data: 13 cells
• PhWT-Flag 2013-06-22, 17 cells
• PhML-Flag 2013-06-22, 34 cells
• Ph and Pc doubles in ML, 2013-08-23 28 cells
• Ph and Pc doubles in WT, 2013-08-23 23 cells
• 2014-01-29 end-PH in PhML: 36 cells
• 2014-01-29 end-PH in PhWT: 55 cells
• 2014-03-13 Pc in S2 cells: 29 cells

Project 2

• see post

Also to do

• Laundry
• deposit check

Mentoring

• discuss probe making protocol with BB
• recommend RNA gel to diagnosis possible degradation
• failure to degrade RNA after RT may also be a problem, this step was skipped

Travel Plans

• booked hotel for Fly Meeting (my card)
• booked flights for Fly Meeting (my card)
• looking in to registration for CSHL Genomes meeting

10:00 am – 12:30 am

K99

• Finished rewriting Research Plan
• Finished editing other sundry components of application
• sent final application and checklist to Judy’s office

9:50 am
(delayed start shoveling out driveway and bikepath to backdoor).

K99

• Revising

Communication

• updated description of researchers and projects for Biophysics reviews for XZ

10:00 am

Meetings

• group meeting (see notes)
• journal club (see notes)
• quick discussion with XZ about reviews

K99

• completed online training in conflict of interest reporting and management
• filed required conflict of interest report paperwork
• Have comments on Research Proposal from XZ, AS, and AP to incorporate

Project 2

• see protected post

DNA sequencing using polymerase substrate-binding kinetics (Nature Communications)

Intro

illumina sequences

• build clusters. deblock of 3′ site, adding of base. remove base. Deblock site. add base

ion-torrent –

• natural nucleotides – faster, no extra chemistry, cheaper, longer read lengths, do require oil emulsions. faster run time. (10-20x more expensive)

New method

• measure signal from fluorescent polymerase bound, use difference in kinetics of correct vs incorrect base to measure sequence
• up to 3 fold difference in fluorescence at high salt (in bulk)
• up to 5 fold difference in fluorescence observed on glass
• homopolymer calling: kinetic difference and total fluorescence combined help distinguish homopolymers
  • better for 1 vs 2 vs 3 than 3 vs 4 (went fast)
• 44 rounds of flowing bases, sequence bases
• sequence phage genome (clip reads at 20bp). 95% accuracy in non-homopolymeric regions, 90% accuracy in homopolymeric regions.

for improvement

• better engineered enzyme
• improve base calling algorithm (correct for local sequence context)
• calibration sequences – normalize for sequence size
• surface modifications (does labeled polymerase accumulate stuck to the surface?)
• better fluidic mixing and flow?