EST Sequencing: QIAGEN Prepping

General Description: The DH10Bä E. coli host cells containing the plasmid with DNA insert of interest have been incubated and pelleted in 96-deep-well blocks in preparation for QIAGENâ prepping. QIAGENâ prepping prepares the source DNA for sequencing reactions by alkaline lysis of bacterial cultures, clearing lysates and isopropanol/ethanol precipitation of DNA. This protocol describes the procedures for alkaline lysis and clearing lysates/DNA precipitation using the QIAGENâ R.E.A.L. Prep 96 procedure.

Materials & Reagents:

Material / Reagent	Vendor	Stock #
QIAGENâ plasmid mini kit	Qiagen	12125
RNase A 100mg/ml (500 ml)	N/A	N/A
R1 Resuspension Buffer	N/A	N/A
R2 Lysis Buffer	N/A	N/A
R3 Neutralization Buffer	N/A	N/A
QIAvac 96 vacuum manifold	Qiagen	19504
96-deep-well blocks	Qiagen	19573
MATRIX Impactâ cordless multichannel pipet	Matrix Technologies	6004
Plate sealers	Edge Biosystems, Inc.	48461
Fisher Vortex-Genie 2ä Mixer	Fisher Scientific	12-812
Water bath	N/A	N/A
Autoclaved deionized and distilled water (ddH2O)	Prepared at Genome Center	N/A
Isopropanol	N/A	N/A
70% Ethanol (4 °C)	N/A	N/A
Fan	N/A	N/A
Ice	N/A	N/A
Jouan centrifuge CR 412	N/A	N/A

Procedure
Taken in modified form from QIAGENâ R.E.A.L. Prep 96 Plasmid Protocol, R.E.A.L.â Prep 96 Plasmid Kit Handbook 09/2000

Alkaline Lysis of Bacterial Cultures

1. If there is no R1 Buffer with RNase A added already in the refrigerator, then add 500 ml of 100 mg/ml RNase A to R1 Buffer using pipette.
2. Add some of the R1 Buffer back to the capsule and add back to R1 to make sure all of RNase A is used.
3. Mix R1 thoroughly by swirling briskly.
4. Be sure to add a check for RNase A added and date and initial the R1 bottle.
5. Set two MATRIX pipettes to fill 1200ml and dispense 300 ml of R1 Buffer into each well of 96-deep-well transfer blocks.
6. Vortex each deep-well transfer block on high until bacterial pellets are re-suspended.
7. Remove covers.
8. Set MATRIX pipette to fill 1200 ml and dispense 300 ml of R2 Lysis Buffer into each well of deep-well transfer blocks.
9. Tape seal deep-well blocks and invert ten times, using tape pad as a backing. (Note: Repeat this step once for every two blocks.)
10. Set the timer and wait for 5 minutes.
11. While waiting, plug in the water bath, set to high and make sure that the water is filled approximately halfway.
12. Prepare to fill 1200 ml and dispense 300 ml of R3 Neutralization Buffer.
13. At the end of 5 minutes, dispense 300 ml of R3 Buffer into each well of deep-well transfer blocks.
14. Invert ten times, following procedure described above with the R2 Buffer.
15. Place deep-well transfer blocks into boiling hot water bath for 5 minutes.
16. While waiting for hot water bath, place some ice in a tray.
17. At the end of 5 minutes, take deep-well transfer blocks out of hot water and pack in ice or an ice water bath if rapid cooling is desired. At this point, deep-well transfer blocks may be left on ice for a few hours if need be.

Clearing Lysates and Isopropanol/Ethanol Precipitation of DNA

1. Prepare 4 new deep-well blocks by adding 600 ml of isopropanol to each well.
2. Place a QIAvac 96 vacuum manifold in correct orientation on top of blocks.
3. When 20 minutes have expired, take deep-well transfer blocks out of ice and bring to bench to place on paper towels.
4. Label new deep-well blocks with plate numbers and fill with 600 ml isopropanol.
5. Then proceed to transfer the contents of the deep-well transfer blocks to the corresponding wells on the QIAvac 96 vacuum manifold/isopropanol block.
6. Make sure that entire well volume is taken up before transferring.
7. After transferring to new deep-well blocks, place blocks in QIAvac 96 vacuum manifold, ensuring that the seal is tight.
8. Turn on vacuum nozzle until volume in each well of QIAvac 96 vacuum manifold has been drawn down into corresponding well filled with isopropanol.
9. When done with all blocks, carefully lift each block out of QIAvac 96 vacuum manifold and throw away.
10. Tape seal only one block at a time and invert three times (Ensure a complete seal with tape and invert quickly or isopropanol will loosen hold on tape).
11. Spin blocks in the centrifuge for 20 minutes, 4 °C at 3200 rpm.
12. Dump media out gently and pat on paper towels to remove any excess media.
13. Retrieve blocks and add 300 m l of cold 70% ethanol into each well.
14. Spin in the centrifuge for 3 minutes, 4 ° C at 3200 rpm.
15. Repeat dumping and patting procedure as described above.
16. Place in front of a low volume desktop fan to air dry.
17. When blocks are dry, dispense 50 m l of autoclaved ddH₂O into each well and tape seal.
18. Vortex for ~ 30 seconds on high.
19. Store at 4 °C until needed for running reactions.