User:Jmcconnell

Jess is a lab technician for iGEM.

Overview

The 2009 DNA part kit will be comprised of around 1000 parts contained on 3 384-well plates. We need to create 200 copies of the kit, producing a total of 600 384-well plates. The parts are contained on plasmids in cultures of E. coli frozen in glycerol stocks in 96-deep-well (96d) plates in -80 freezers at the registry.

We are going to inoculate 96d plates from the freezer stocks, incubate them, then take them across the street for mini-prepping in 6 groups of 4, for a total of 24 96d plates (2 copies of each unique plate).

For every set of 4 unique 96d plates that we get back (once per week) we will use EP 5075 to compose the mini-prepped DNA into 4 copies of a unique 384-well intermediate source plate (ISP). We will use these 4 copies of the ISP (at 200 ul per well and 150 ul expected transfer capacity per well/3 ul per well to destination kit plates= 50 copies per ISP * 4= 200 copies for 200 kit plates) with the PMP to transfer 3.0 uL of DNA into 200 384-well kit plates. This cycle will take place 3 times, over the course of 3 weeks, producing an uncollated set of 600 384-well plates for the 2009 DNA part kits. We would like to factor in drying time (roughly 1 week) so that we do not have to spend a large amount of time at the end waiting for kit plates to dry.

General Schedule

Mondays

Streak out existing stock on an appropriate plate or transform 2008 incoming DNA into Top10 cells, spread on appropriate plate. Grow up overnight. (allow 16-17 hours for growth) 192 samples

Tuesdays

Use pipette to pick single colony from previous day's growth; do this with 96 different colonies to create a unique 96-well staging plate (50 ul water in each well). Use Pin Tool to replicate this staging plate into 96-well plates for mini-prepping (2), 96-well plates for glycerols (2), and (for the new parts from 2008) previously-ordered 96-well plates for antibiotic testing (4).

Ultimately 192 unique colonies will be used to create 2 96-well staging plates are copied to yield 16 96-well plates for various purposes. Incubate 12 of these plates for mini-prepping and antibiotic testing overnight (allow 15 hrs); refrigerate the remaining 4 glycerol plates to incubate later.

Wednesdays

Begin incubating the glycerol plates (4 in total, 2 from each 96-well staging plate), allow 12 hrs. Take out mini-prep plates and AB testing plates (12 in total; 6 from each 96-well staging plate). Spin down, image growth, upload pics to wiki. Send mini-prep plates over to Tsultrim (2 copies of 2 different staging plates)

Post mini-prep: 2 unique plates of purified DNA come back to us for inclusion in 384 well Intermediate Source Plate-- the other 2 plates will be used for sequencing + restriction digest (confirm).

Later in the day-- Streak out existing stock on an appropriate plate or transform 2008 incoming DNA into Top10 cells, spread on appropriate plate. Grow up overnight. (allow 16-17 hours for growth) -- 192 samples

Remove glycerol plates from incubator, store in freezer.

Thursdays

Use pipette to pick single colony from previous day's growth and do this with 96 different colonies to create a unique 96-well staging plate (50 ul water in each well). Use Pin Tool to replicate this staging plate into 96-well plates for mini-prepping (2), and 96-well plates for glycerols (2), and previously-ordered 96-well plates for antibiotic testing (4).

Incubate 12 of these plates for mini-prepping and antibiotic testing overnight (allow 15 hrs); refrigerate the remaining 4 glycerol plates to incubate later.

Fridays

Begin incubating the glycerol plates (4 in total, 2 from each 96-well staging plate), early AM (space permitting) allow 12 hours. Remove mini-prep plates and AB testing plates from incubator (12 in total; 6 from each 96-well staging plate). Spin them down, image them, upload pics to wiki. Send mini-prep plates over to Tsultrim (2 copies of 2 different staging plates)