This list follows an example project through AGTC. This particular project is a gene-sequencing project involving the isolation and analysis of interesting clones from a whole-genome library of shotgun clones. AGTC's flexible menu of Schedules (link to Schedules page) can accommodate many other types of molecular-biology projects, and allows the researcher to submit samples and retrieve data at nearly any step in the process.

• START: Client/PI registers project with consults with AGTC


• Client uses Spex GenoGrinder to Isolate Genomic DNA from organism -Schedule19


• Client shears DNA in HydroShear and runs gel to check shearing -Schedule19


• Client ligates sheared fragments (with or without adapters) into Vector Plasmid/Cosmid (Client's Lab)


• Client Transforms clones into Bacteria (Client's Lab)
  
• Client: Spread and Grow Bacteria on Gentix QTrays (See AGTC Supplies Catalog)


• AGTC recommends Client run preliminary digest gel of randomly picked clones to ensure library does not contain inordinate number of empty or unwanted clone types (Client's Lab)


• PI creates project on AGTC online LIMS system at http://genome.kbrin.uky.edu/cgi-bin/AGTC/login.pl - Alternate time of project creation might be after Re-arraying step


• Client Submits QTrays to AGTC for processing through Schedule 12


• AGTC picks Colonies with QPix2 robot into 96 or 384-well Source Plates Containing Freezer Media to make Library (Schedule12 cont)


• At Client's request, AGTC uses QPix2 robot to create a MacroArray Grid of Library onto Nylon membranes (Schedule 16)


• Client Probes Macroarray Filters to look for interesting target clones. If radiation is used, Client provides .tiff of autoradiogram. If flourescent tags are used, then macroarray may be read using AGTC's Flouroimager 595 (Schedule 19)


• .tiff image is imported into AGTC's Incogen HDFR softwareand interesting clones are identified and their locations in the library are mapped (Schedule 19)


• AGTC then Re-arrays interesting clones into contracted to-be-sequenced library using Biomek FX (Schedule 21) AGTC LIMS tracks mother-daughter relationship of samples throughout process.


• AGTC grows clones to be sequenced in GeneMachines HiGro or Tomtec incubator (continuation of Schedule 12)


• Incubated bacteria are miniprepped on Biomek FX robot using appropriate kits for plasmids/cosmids/etc. (Schedule12 cont)


• AGTC Quantitates DNA minipreps using SybrGold stain on FlourImager595. AGTC also checks clone integrity by gel electrophoresis. Images and data is uploaded to Client's LIMS folder for review and future use (Schedule 12 complete)


• AGTC Performs Sequencing Sanger Reaction using ABI BigDye or Beckman DTCS chemistry on in-house thermalcyclers (Schedule7 begins)


• Cycle Sequencing reactions are cleaned up on Biomek FX robot (Schedule7 cont)


• AGTC Registers Sequencing Plate (aka Reaction Plate) on AGTC Online LIMS System. Client can track status of samples through AGTC in real-time via LIMS


• AGTC Staff run Sequence Plates on Beckman Coulter CEQ8000 or ABI sequencers (Schedule7 cont)


• Sequence Results: Analysis Script Quality Report is automatically emailed to the User and PI (Schedule7 complete)


• PI's and Users can access and download all sequencing results in their projects from the archive of CEQdata. Each PI's folder is password-protected. Web address is http://genome.kbrin.uky.edu/CEQdata/


• User downloads files to computer and views with favorite downstream software


• User analyzes sequence data or assembles contigs in third-party downstream software, such as VwectorNTI, available for free at http://www.biology.uky.edu/vectornti/


• Other bioinformatic services provided free of charge by AGTC staff. END