MATERIALS:

4-Beckman Deepwell Blocks (96 well) 250ml AC grad.cylinder

8- Dynex v-bottom plates (96 well) 500ml AC Erlenmeyer

4-Costar Deepwell 4 boxes AC white tips

12 gas permeable seals 1 box AC yellow tips

1 foil seal 30ml disposable LL syringe

TB (Terrific Broth) w/o Salts Finnpipette w/ 8-channel adapter

10x salts 50ml Eppendorf syringe w/ adapter

Ampicillin (2-ml vials) Eppendorf Repeat Pipettor

100% Glycerol TB V-groove reservoir

• Autoclaved (AC) glassware kept in cabinet at the end of the first bench in the main lab.

• Note: Be sure to carefully observe sterile technique throughout procedure.

1. Remove and log out stock plate. (Stock plates are stored in the –80 upright freezer in the main lab, record date and initials in space provided for that stock plate on the log out sheet attached to the front door of freezer; stock plates are stored on the 3^rd shelf, remove stock plate from the left, when inoculation is complete, return stock plate to the right side and store over night. Next day, dispose of stock plate in hazardous waste disposal bag, after noting acceptable growth.)
2. Thaw approximately ½ hour on Titer Plate Shaker. (Shaker closest to the sink, good speed is 4. Shaker further from the sink, good speed is about 6.)

(Work under the laminar flow hood and observe sterile technique.)

1. Summary and worksheets are kept in the respective library notebook. Pull the notebook for the library that is being prepped. Pull 1^st 4 worksheets (without results) after the last worksheet with completed results. Fill in the summary sheet (initials and plate name, record date when mini-prep is completed.)
2. Gather materials and place under hood:

• 4-Beckman Deepwell Blocks (96 well)

• 8- Dynex v-bottom plates (96 well)
• 12 gas permeable seals

• TB (Terrific Broth) w/o Salts (Broth and salts are kept in media cabinet in Hydra Lab)
• 10x salts
• 1.5ml 100mg/ml Ampicillin (Amp is kept in –20 upright freezer in the main lab.)
• 100% Glycerol

• 250ml AC grad.cylinder
• 500ml AC Erlenmeyer
• 4 boxes AC white tips
• 1 box AC yellow tips
• 30ml disposable LL syringe
• Finpipette w/ 8-channel adapter
• 50ml Eppendorf syringe w/ adapter
• Eppendorf Repeat Pipettor
• Disposable reagent reservoir

1. Labeling: Label 1 Deepwell and 2 Dynex v-bottoms, per sample, with sample number, initials and date. Dynex v-bottom plates are labeled on the left and front side of the plate with a paint pen and label Beckman Deepwell on front of block with tape and paint pen.
2. Prepare TB broth for Beckman Deepwell and Dynex v-bottom plates:

• To 900ml of TB broth, add 100 ml of 10X salts and 1.5ml 100mg/ml final concentration 0.15mg/ml), swirl vigorously.

1. Assemble Eppendorf repeat pipettor: attach adapter first, than attach 30ml syringe and set to 1.5ml. Fill disposable reagent reservoir with fortified TB broth and fill the 4 Beckman Deepwell’s with 1.5ml of Broth in each well. Empty excess broth in reservoir into waste.
2. Prepare freezing medium: To 200ml fortified TB broth in 500ml Erlenmeyer flask, add 14ml 100% Glycerol (final concentration 6.5% ), swirl vigorously. Fill disposable reagent reservoir with freezing medium and fill the 8 Dynex v-bottoms with 150ul each well. (To clean reagent reservoir and syringe, rinse both with distilled water into waste and again with 70 % EtOH and let dry under hood.)

1. Remove stock from Titer Plate Shaker to hood, be careful not to splash and cross contaminate wells. Carefully remove aluminum foil and discard into hazardous waste bag.
2. Calibrate the Finnpipette: 8 tip X 10.00ul head.

• To change head lift cover to left of display, grasp tip head and pull straight off pipettor.

• To calibrate, open cover and than close cover, depress + or – to set, e.g. 8(tip) 10.00ul (head). Depress set. "Calibrate" will flash, depress aspirator trigger all way down and hold.
• Mode chooses stepper option, which dispenses in increments or pipette option. Choose stepper, depress set than + or – to set 1ul.
• Depress set again to select 5, (stepper will dispense 1ul 5 times).
• Depressing trigger completely blows out remaining sample and re-calibrates (must keep trigger depressed long enough to reset or recalibrate). Release to the first stop, aspirate, each time trigger is depressed to first stop, aliquot is dispensed as per protocol.

1. Stock plates contain 384 unique samples. Use the 8 tip Finnpipette to inoculate blocks and Dynex v-bottoms in this order:

• 1^st set = wells 1-12 (top row) = 1^st Beckman Deepwell and 2 Dynex v-bottoms.
• 2^nd set = wells 13-24 (top row) = 2^nd Beckman Deepwell and 2 Dynex v-bottoms.
• 3^rd set = wells 1-12 (2^nd row from top) = 3^rd Beckman Deepwell and 2 Dynex v-bottoms.
• 4^th set = wells 13-24 (2^nd row from top) = 4^th Beckman Deepwell and 2 Dynex v-bottoms.

1. Aspirate 5ul, dispense 1^st 1ul aliquot back into original sample, 2^nd 1ul aliquot into 1^st Dynex v-bottom, 3^rd 1ul aliquot into 2^nd Dynex v-bottom and 4^th 1ul aliquot into the Deepwell; last ul aliquot stays in tip and tips are discarded. Repeat for all samples.

1. As plates are completed, cover with gas permeable seals (tabs align to the right edge of the blocks and plates when blocks/plates are facing frontward). Incubate Beckman Deepwell blocks at 37C, 250 RPM for 20 hours and Dynex v-bottom plates at 37C, 250 RPM for 16 hours in HiGro incubator. HiGro: Be sure each empty chamber has an empty plate in the top slot and the bottom slot and chamber is fully closed before starting agitation, this holds rack in place. Also, as Rpm’s are increased at start-up, listen for any noise and be sure it is operating quietly and smoothly before walking away. Always a good idea to leave a note on top identifying samples in the chamber. (Just in case someone turns it off and removes their samples and leave yours.)

1. At 16 hours pull Dynex v-bottoms from Hi-Gro. To retrieve plates, slowly lower Rpm’s to stop and than power off. Be very careful not to invert or set plates down to hard. Do not want any splashing that would cross contaminate wells when gas permeable seals are removed.
2. Observe each well in Dynex v-bottom plates and record any wells that have none or light growth. Carefully remove gas permeable seals under the laminar flow hood and replace with pre-cut Robbins foil seals and transfer to –80 freezers (One set into upright –80 freezer and one set in newer chest –80 freezer in main lab). Samples are stored beginning with the lowest number on bottom and highest number on top in a vertical stack in cassette drawer (samples go front to back on left and front to back on right as left is full.) Record initials and date of stock sample plate placed into the freezer on the sample log sheet attached to the front of the freezer door and/or the top of the freezer chest.
3. At 20 hours pull Beckman Deepwell blocks and centrifuge for 5 minutes @ 3000 RPM, (Recall 31, enter, start.) (Samples in at 1pm are out at 9 am the following morning.)
4. Place Saran wrap on bench to avoid contamination and place double thickness paper towels over the Saran wrap. Set waste box next to the saran wrap/paper towel. After centrifuging, remove gas permeable seals and carefully pour off the supernatant into waste box and set inverted Deepwell blocks onto paper towels, drain for a minute, move over to a dry spot on the towels and let drain for another minute. Send waste box to autoclave.

Note: To decant supernatant, use one up and over motion to decant into waste, give one good shake and set inverted block onto paper towel to drain. Be sure to quickly invert blocks so as not to cross contaminate and carefully so as not to splash back into the Deepwell. DO NOT TURN THE BLOCK TOPSIDE, AFTER DECANTING, OR BEFORE DRAINING is complete and the top of the block is dry, TO AVOID CROSS CONTAMINATION.

HYDRA PROTOCOLS:

• Fill fresh water carboy to full.
• Rinse reservoir with ddistilled water (3X) before filling with appropriate reagent.
• Double check blocks and plates are positioned/labeled correctly.

• Blocks/Plates stick to needle plate, be sure to hold when plate rises.

• Be sure to keep stock chemicals ready. (TE-buffer, SDS/NaOH, KOAc)

• When run is complete rinse reservoir with distilled water followed by 70% EtOH, let dry.
• NEVER LEAVE HYDRA UNATTENDED WHILE RUNNING A PROTOCOL.

Path to Hydra Protocols:

C:

Program Files

Plate Positioner

User Protocols

High Throughput

3700 Run Preparation

Cycle Sequence Protocols

DNA Precipitation

70% EtOH Addition

Resuspend Template DNA 1 block

Resuspend Template DNA 4 blocks

Transfer Resuspended Template DNA 1 Block

Transfer Resuspended Template DNA 4 Blocks

G-50 Preparation Clean-Up

Template Isolation

Supernatant Transfer 1 block

Supernatant Transfer 4 blocks

Template DNA Isolation Solutions Addition 1 block

Template DNA Isolation Solutions Addition 4 blocks

Washes

Standard Wash

Fill In Between Runs

Quick Quleen 1

• CAUTION: These next steps can be tricky if the correct protocols are not carefully selected. Be sure the correct protocol is selected before running Hydra. Exercise special care at this step.

1. To 100ml TE-buffer in an autoclave graduated cylinder, (record lot # of buffer in notebook and summary sheets), add 0.5ml Rnase A (10mg/ml kept in –20 freezer), cover with parafilm and mix well. Place Lysis/Buffer reservoir in appropriate nest, pour all TE-Rnase A solution into reservoir.
2. Set up Hydra to add 200ul TE-RNase into sample well of each Beckman Deepwell x 4:

1. Hydra should be in the stand-by position: 290ul water in syringes, tips in water

MESSAGE: PLEASE LEAVE HYDRA AS IS. PRESS OK WHEN READY TO RUN PROTOCOL. SELECT OK.

2. Hydra will automatically empty syringes, wash and prepare for run.

MESSAGE: RUN COMPLETED, DATE AND TIME. SELECT OK.

3. Blank screen appears; Select: File > Run >Select Hydra Protocol: TEMPLATE DNA ISOLATION SOLUTIONS ADDITION 4 BLOCK. Protocol will pop up, check to be sure it is the program you selected and read the description of the source plate and the destination plate to be sure you have the correct plates in the correct nest, e.g., for lysis buffers, reservoir will be the v-groove reservoir and destination will be the Beckman Deepwell block. SELECT RUN.

MESSAGE: THE SYRINGES MUST BE EMPTY BEFORE RUNNING PROTOCOL. DO YOU NEED TO EMPTY SYRINGES. SELECT NO IF THE SYRINGES ARE EMPTY. (LED is displayed on the front of the Hydra, if it shows 0.0ul, than syringes are empty. If it says empty or shows volume SELECT YES and follow the onscreen instructions.)

4. Follow directions for block/plate placement. SELECT OK, program begins. As each block finishes, Hydra will ask for the next block. Remove 1^st block, set to side, set 2^nd block in nest and repeat for remaining blocks.
5. At the end of the run, remove final block, remove Lysis/Buffer reservoir, drain reservoir and rinse with distilled water into the sink, rinse with ddwater and finally with 70% EtOH, set in drain to dry. Hydra will automatically wash needles and than ask if you are done or want to run another program. If done, SELECT YES. Hydra will fill syringes with water and standby for next user. If not done, SELECT CANCEL and Hydra will go back to the blank screen and wait for a program to be selected.

1. Cover blocks with Deepwell lids and set on Titer Plate Shaker for 45-60 minutes. (7 is a good speed, want good dissolution of the pellets.)
2. Place rubber bands on samples (vertically and horizontally, to stabilize blocks.)
3. Remove from shaker.

Bacterial lysis:

[SDS/NaOH lyses the bacteria and denatures macromolecules.]

4. Hydra: Same protocol as above beginning with 2A, fill reservoir with SDS/NaOH. Protocol will transfer 200ul SDS/NaOH from v-groove reservoir into each well of the 1^st block than prompt you for the next block, etc. Return to Titer Plate Shaker and shake for 45-60 minutes (6 is a good speed; Don’t want to shake to hard as DNA could be severed or broken.)

Aggregation of bacterial debris:

[KOAc aggregates denatured macromolecules and neutralizes the solution.]

5. Hydra: Same protocol as above beginning with 2A, fill reservoir with 3M KOAc pH 5. Protocol will transfer 200ul 3M KOAc pH 5 from v-groove reservoir into each well of the 1^st block than prompt you for the next block, etc. Cover 1^ST BLOCK with Acetate film. (Wait to fill the 2^nd block until 1^st block is complete, etc.)
6. Mix on vortex: Hold with 2 index fingers to create a band across the top; place block on shaker while depressing pad all the way down to avoid movement and release pressure to start vortex. Goal: even vortexing within all wells (no splashing or churning) so that you can see the black vortex pad through each well. Vortex 3 minutes, at 1.5 minutes rotate block and continue vortexing for 1.5 minutes. (USE TIMER.)
7. As each block is finished, place on Titer Plate Shaker (ca. speed 2). When all blocks are complete, remove and transfer to 37C, 250-RPM shaker for 20 minutes.
8. Remove from 37C, 250-RPM shaker (acetate cover stays on) and keep at -80 for a minimum of 8 hours.

1. Remove samples from –80; remove acetate cover and place under hood to thaw (approximately 2.5-3 hours; want pellet and supernatant thawed). Straddle on plate tops to get a good flow around and under plates for more even thawing.
2. When plates are fully thawed, centrifuge for 45 minutes at 3850 RPM (program 35).
3. Transfer supernatant. Hydra Protocol: Beginning with step 2A (Hydra Protocols) above SELECT SUPERNATANT TRANSFER 4 BLOCKS at step 2C. Check to be sure it is the program selected and read description of source plate and destination plate to be sure the correct plates is in the correct nest, e.g., for supernatant transfer, reservoir will be the Beckman Deepwell Block and destination will be the Costar Deepwell Block.
4. Wash bacterial debris out of Beckman block as soon as protocol is complete, to avoid crusty build-up, rinse plates with warm/hot tap water to remove debris, rinse with dwater and finally with ddwater, set inverted on cart to dry.

• CAUTION: This is a clean up and recovery step, use good technique and be careful to not contaminate and/or cross contaminate plates.

1. To transferred supernatant in Costar blocks, add 500ul of 95% EtOH to each well with Eppendorf repeat pipettor (need adapter, 30ml syringe and v-groove distilled water/ETOH reservoir).
2. Centrifuge for 30 minutes at 3850 RPM (Program 34).

Gently decant (turn over and pour, no hard shake, it is possible to loose DNA) supernatant into sink, place inverted Costar onto Saran/paper towels. (DO NOT TURN THE BLOCK TOPSIDE AFTER DECANTING, OR BEFORE DRAINING TO AVOID CROSS CONTAMINATION.)

3. Drain about a minute, than move to a dry portion of paper towel to continue drying and to dry top of block.
4. Add 250ul of 70% EtOH to each well. Hydra Protocol beginning with step 2A above, SELECT 70% ETOH ADDTION at step 2C. Check to be sure it is the program you selected and read description of source plate and destination plate to be sure you have the correct plates in the correct nest, e.g., for 70% EtOH Addition, reservoir will be the V-groove reservoir and destination will be the Costar Deepwell block
5. Immediately centrifuge 15 minutes @ 3850 RPM (program 33).
6. Again decant supernatant into sink, place inverted Costar onto Saran/paper towels. (DO NOT TURN THE BLOCK TOPSIDE AFTER DECANTING, OR BEFORE DRAINING TO AVOID CROSS CONTAMINATION.)
7. Dry (small incubator, behind the door with message board), at 37C for 30 minutes (No longer than 1 hour)
8. Add 150ul ddistilled water (Use bottled sterile water) to each well. Hydra Protocol beginning with step 2A above SELECT RESUSPEND TEMPLATE DNA 4 BLOCKS at step 2C. Check to be sure it is the program you selected and read description of source plate and destination plate to be sure you have the correct plates in the correct nest, e.g., for Resuspend Template DNA 4 Blocks, reservoir will be the V-groove reservoir and destination will be the Costar Deepwell block
9. Cover with Foil and return to Titer Plate Shaker for 15 minutes (ca. speed 7).
10. Transfer 150ul DNA to Dynex v-bottom plate. Hydra Protocol: Set up Protocol beginning with step 2A above SELECT TRANSFER RESUSPENDED TEMPLATE DNA 4 BLOCKS at step 2C. Check to be sure it is the program you selected and read description of source plate and destination plate to be sure you have the correct plates in the correct nest, e.g., Transfer Resuspended Template DNA 4Blocks, reservoir will be the Costar Block and destination will be the Dynex v-bottom (96 well).
11. Store at –20C and/or quantify DNA with Fluorometer downstairs.
12. Record date mini-prep is completed on summary sheet.

• ALWAYS leave system washed, needles full of water and tips in water, while system waits for next user. Under normal protocol circumstances, this is done automatically at the end of each run.

1. Standard Wash: Hydra Protocol beginning with step 2A above, SELECT STANDARD WASH at step 2C. Check to be sure it is the program you selected. Hydra automatically washes 3 times and finishes. At this point needles need to be filled with water and tips left in water or select another protocol.
2. Fill In Between Runs: Set up Protocol beginning with step 2A above, SELECT FILL IN BETWEEN RUNS at step 2C. Check to be sure it is the program you selected. Hydra fills needles with 290ul of water and rests with tips in water ready for the next user.