Difference between revisions of "User:Jmcconnell"
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Jess is a lab technician for iGEM. | Jess is a lab technician for iGEM. | ||
| − | Overview | + | '''Overview''' |
The 2009 DNA part kit will be comprised of around 1000 parts contained on 3 384-well plates. We need to fabricate 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. | The 2009 DNA part kit will be comprised of around 1000 parts contained on 3 384-well plates. We need to fabricate 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. | ||
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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. | 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 | + | '''General Schedule''' |
''Mondays:'' | ''Mondays:'' | ||
Revision as of 21:41, 27 March 2009
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 fabricate 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 miniprepping 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 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)
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).
We now have a total of 4 unique plates of purified DNA with which to create 384 well Intermediate Source Plate.