Isolation of DNA templates

Picking and Growing

1. We pick colonies using a Flexys automated picker from Genomic Solutions
2. The colonies are grown in deep-well blocks (96 wells) containing 1.5ml of 2xYT media (100µg/ml amp) per well. The blocks are covered with either foil or Breath-easy film covers, incubated at 37ºC, and shaken at 225rpms for 16 to 20 hours.
3. We make a freezer stock for each block by combining 100µl of cells with 100µl of sterile 50% glycerol into a sterile, freezer-safe plate. Cover the plate and store at -80ºC.
4. Centrifuge the deep-well block at 3250rpm, 4ºC, for 10 minutes to pellet the cells.
5. Dump the supernatant and leave block upside down for 10 minutes to dry. The supernatant should be sterilized before being thrown out.

 

Plasmid Prep

1. We use a Biomek 2000 automated robot from Beckman to prep the blocks.
2. The pellets are resuspended by pipetting up and down 10 times with 200µl of Solution I.
3. Then, 200µl of Solution II is added and mixed by pipetting up and down 10 times.
4. Finally, 200µl of Solution III is added to each well. It is not necessary to mix.
5. We cover each block with a Thermalseal plate cover and store the blocks at -20ºC, at least overnight.

 

 Ethanol Precipitation

1. Let the blocks thaw at room temperature for approximately 1 hour.
2. Centrifuge at 3250rpm, 4ºC, for 1 hour.
3. Transfer 160µl - 2x to a clean block, making sure not to transfer the pellet.
4. Add 1ml of room temperature 95% alcohol to each well.
5. Let the blocks precipitate at -80ºC for 1 hour.
6. Centrifuge at 3250rpm, 4ºC, for 1 hour.
7. Immediately remove the covers from the blocks, invert each block over a container that can catch the waste, set (still upside down) on clean paper towels for only 5-10 seconds, and turn each block right side up.
8. Add 400µl of cold 70% alcohol per well to wash the pellet.
9. Centrifuge at 3250rpm, 4ºC, for 15 minutes.
10. Dump the waste by inverting, as in step #7.
11. Add 400µl of cold 70% alcohol per well for a second wash.
12. Centrifuge at 3250rpm, 4ºC, for 15 minutes.
13. Dump the waste as before, but this time lightly tap each block on the paper towels to help remove excess liquid.
14. Dry the blocks in a vacuum oven heated to approximately 85ºC. The drying time usually ranges from 40 minutes to 1 hour, depending on the amount of excess liquid in each well.
15. Once the blocks have dried, add 100µl of DIUF water per well.
16. Cover with a fresh Thermal seal, vortex well, quick spin to pull the liquid to the bottom of the wells, and store at -20 ºC.

 

 Solution I: 100ml

• 93ml milliQ water
• 2ml 0.5M EDTA
• 5ml 1M Tris-HCl
• 0.004mg RNAse A
• 8µl RNAse T

note: make this solution fresh each time.

 

 Solution II: 100ml

• 80ml milliQ water
• 10ml 10% SDS
• 10ml 2N NaOH

 

 Solution III: 100ml

• 100ml 3M NaOAc pH 4.8

 

 notes

• Each 100ml batch of solution will prep 4 blocks
• Method adapted from Bruce Roe's "Biomek 2000 Double Stranded DNA Isolation of DNA Sequencing Templates" protocol, which can be found here.