Version 2.1
Supplies:
- Whatman Unifilter (Whatman, Part#. 7700-1801).
- Whatman Uniplate (Whatman, Part#. 7701-1800).
Reagents:
- GET/RNase working solution for 4X96 preps (store on ice):
80 ml GET
1.2 ml RNase A (10 mg/ml)
- 1%SDS/0.2N NaOH working solution for 4X96 preps (store at room temp):
0.8 g NaOH
90 ml sterile water
10 ml 10% SDS
Background:
This protocol was adapted from the Washington University pUC EST template prep protocol. (http://genome.wustl.edu/gsc/Protocols/pucprep.shtml)
Protocol:
- Remove 96-well blocks from shaker after 24-hour growth. Centrifuge block in tabletop centrifuge in equipment room for 8 minutes at 2700 rpm. Remove and inspect for solid pellet. If supernatant is not clear, repeat.
- Remove taped lids and add 100 m
l cold GET/RNase to each well using repeater pipette. Tape block closed with 3M tape and secure block in vortex, shake for 2-5 minutes.
- Remove tape from block and add 100 m
l cold GET/RNase to each well. Seal and repeat shaking on vortex.
- Check that the pellet has been completely resuspended. If cells clumps are still visible, resuspend using 8-channel pipette. Store block on ice between steps.
- Add 200 m
l 1%SDS/0.2N NaOH to each well. Seal with 3M tape and rock block 20 times. If wells do not clear immediately, incubate for 5 minutes at room temperature.
- Inspect for good lysis.
- Add 200ul 3M KAc pH 5.5. Seal block and invert 5-7 times. Incubate 10 minutes in ice water bath.
- Centrifuge 15 minutes at 4000 rpm.
- While spinning, fill collection plates with 330 m
l cold 100% EtOH. Label collection plates with plate number and quadrant number and place filter plate on top. Store on ice until use.
- Remove tape from plate and inspect for clear supernatant. If supernatant is not completely clear, repeat spin.
- Remove tape and transfer 200 m
l of supernatant to filter block using 8-channel pipette.
- Centrifuge 15 minutes at 4000 rpm.
- Inspect the filter blocks to ensure supernatant has gone though. Dump off ethanol from collection plate and place filter plate in sink.
- Working quickly, ad 250 m
l 70% ethanol to the receiver plate (direct fluid to side of wells so as not to disturb the pellet).
- Dump off ethanol and drain upside down for 5 minutes. Dry in SpeedVac for 5 minutes. Check for residual ethanol.
- Heat TE in microwave for 15-20 seconds. Using 8-channel pipette, add 40 m
l to each well.
- Read 260:280 on spectrophotometer and print data for notebook.
Stock Solutions:
GET Buffer:
36 g Glucose
80 ml 0.5M EDTA (pH 8.0)
50 ml 2 M Tris-HCl (pH 8.0)
3500 ml deionized water
Stir and adjust volume to 4 L.
Filter sterilize.