Difference between revisions of "User:Jmcconnell"
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Overview
 
Overview
  
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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 3 groups of 4, for a total of 12 96d plates. For every 4 that we get back we will use EP 5075 to compose the miniprepped DNA into 3 original 384-well intermediate source plates (SP) of which we will use 4 copies of each original 384-well intermediate source plate (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) with the PMP to transfer 3.0 uL of DNA into 200 384-well kit plates. This will happen 3 times, producing an uncollated set of the 600 384-well plates for the 2009 DNA part kits.
+
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
 
General Schedule
  
−
Mondays:  
+
''Mondays:''
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Streak out on appropriate plate from existing stock or transform and grow up on appropriate plate overnight (16-17 hours growth)
+
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)
−
Tuesdays:
+
192 samples
−
Pick single colony from previous day's growth and  
+
 
 +
''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
  
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Necessary Supplies
+
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).
  
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96d miniprep cultures
+
Incubate 12 of these plates for mini-prepping and antibiotic testing overnight (allow 15 hrs); refrigerate the remaining 4 glycerol plates to incubate later.
−
Making a fresh culture of one of the 96d freezer (library) plates involves filling each well of the new plate with LB and the particular antibiotics used to select for the part clone in the corresponding well of the freezer plate. Randy can generate a .csv file that instructs the EPM to add the antibiotics for any given freezer plate (antibiotic resistance can also be read manually from the Physical DNA section of the part's entry in the registry).
+
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8 miniprep culture plates can be prepared in one EPM run (2 miniprep cultures need to be made for each freezer plate), leaving room on the EPM stage for two boxes of tips and the reservoir of antibiotics. The volume of antibiotic needed in one run is equal to: 8 plates * 96 wells/plate = 768 wells, * 20 uL / well = 15,360 uL
+
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The EPM will add 20 uL of the correct antibiotic to each well, resulting in a 1:100 dilution. If the antibiotic stocks are normally diluted 1:1000 to obtain a working concentration, then they should be diluted 1:10 when put into the EPM reservoir (not necessarily true for tet: 14ml LB + 6 tet stock in reservoir).
+
−
Prepare 150 mL of LB-amp (768 wells * 180 uL/ well = 138.240 mL)
+
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Pre-fill each well of the 96d plates with with 1980 uL of LB-amp (amp conc: 100 ug/mL)
+
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Use the EPM to do this with method file notlisted.ext, or
+
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Use the plate-filler in the deLong lab to do it
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Fill reservoir 1 with 20 mL chloramphenicol at 350 ug/mL
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Fill reservoir 2 with 20 mL kanamycin at 500 ug/mL
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Fill reservoir 3 with 20 mL tetracycline at 150 ug/mL
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howto: import .csv file(s)?
+
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howto: Make sure each plate is in the correct position on the stage
+
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list: settings in EPM method file: what tip head to use, how long the run will take, etc.
+
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Run the EPM
+
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when finished, replace the covers on each culture plate and store in the refrigerator.
+
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96d glycerols
+
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Library plates 1-8 exist in triplicate and are spread across the two Knight lab and one of the Endy lab -80 freezers for redundancy. Duplicate glycerol stocks of the newer, singular library plates (9-16) contributing to the 2007 parts kits should thus be made when the library plates are thawed to inoculate the miniprep plates.
+
−
Before inoculation, the well of a glycerol plate should consist of 792 uL LB-amp (100 ug/mL amp) 8 uL specific antibiotic.
+
−
384d antibiotic test
+
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All part clones will be tested for correct antibiotic resistance during the inoculation of the miniprep plates by also inoculating a 384d antibiotic test (AB) plate. The wells of the AB plates are grouped into four quadrants such that each well of a 96d library plate corresponds to 4 contiguous wells on the AB plate, with each of the 4 correspondent wells containing a different antibiotic broth.
+
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384d AB plate antibiotic quadrant layout
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1 2 3 4 ...
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A amp chm amp chm
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B tet kan tet kan
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C amp chm amp chm
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D tet kan tet kan
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...
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We can prepare all the AB plates in advance with the EPM.
+
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Use 4 methods, one for each antibiotic.
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100ul EZ Rich broth with appropriate antibiotic in each well.
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100ul * 96 wells = 9600ul of each antibiotic broth per plate * 8 plates per run = 76,800ul of broth per run (20ml per reservoir * 4 reservoirs)
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Typical concentrations and volumes
+
  
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Antibiotics (also see BU protocol)
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''Fridays:''
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antibiotic stock working
+
Begin incubating the glycerol plates (4 in total, 2 from each 96-well staging plate), early AM (space permitting) allow 12 hours.
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ampicillin 100 mg/mL 100 ug/mL
+
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.
−
kanamycin 50 mg/mL 50 ug/mL
+
Send mini-prep plates over to Tsultrim (2 copies of 2 different staging plates)
−
chloramphenicol 25 mg/mL 25 ug/mL
+
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tetracycline 5 mg/mL 15 ug/mL
+
  
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plate well volume total volume manufacturer spec sheet
+
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).  
−
96 ? ? ? ?
+
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96d 2 mL 192 ml ? ?
+
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384 110 uL 42.240 mL nunc 242747
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384d 220 uL 84.480 mL ? ?
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Test Run: 10 April 07
+
  
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Meet Sultrim around noon with 6 384 plates filled with 100 uL dyed dH20 per well (230.4 mL ) and 20 empty 384 well plates. Try filling the empty plates with 3.0 uL from the SPs.
+
We now have a total of 4 unique plates of purified DNA with which to create 384 well Intermediate Source Plate.

Revision as of 21:40, 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.