Materials & Reagents

Materials/Reagents/Equipment	Vendor	Stock Number
Disposables
70% Ethanol	JGI	N/A
Foil Sealers	Beckman	N/A
Reagents
STET-E1	JGI	N/A
50mg/ml Lysozyme	Sigma	N/A
10mg/ml RNaseA	Sigma	N/A
PEG/NaCl	Teknova	N/A
Magnetic Bead Suspension	Seradyn	N/A
Deionized Water	N/A	N/A
Equipment
Multidrop (single plate)	Titertek	N/A
Mutlidrop (20 shallow well plate stacker)	Titertek	N/A
Shallow well Plates	Falcon	N/A
Allegra Centrifuge	Beckmen	N/A
5810 Centrifuge	Eppendorf	N/A
Hydr96 (290ul syringes)	Robbins Scientific	N/A
Mini Orbital Shaker	Bellco	N/A
Kim Towels	Kimberly Clark	N/A
Plate Washers	Biotek	N/A
Seal Roller	Speedball	N/A
Electrapette (125ul, 250ul, 850ul, 1250 ul)	Matrix	N/A
Electrapette tips (250ul, 1250 ul)	Matrix	N/A
Multitube vortexer	VRW	N/A
Freezers (-20C and -80C)	REVCO	N/A
Horizontal Flow Oven	VRW	N/A
370C Incubator	VRW	N/A
CRS platform w/ articulated robotic arm	CRS/JGI	N/A

Procedure

Post Inoculation:
1. After 18 hour incubation, remove Falcon culture plates from the HiGro incubators

2. Track the HiGro incubators and the HiGro cylinders by scanning each barcode label on each plate into the computer database according to the incubator number and cylinder number from which the plate is removed.

3. Record the number of wells in each plate which yielded no growth. If there are more than eight wells which yield no growth in any one plate, dispose of the plate and enter it into the re-inoculation queue.

4. At the PC, scan barcodes and print labels for the fresh transfer plates.

STET Addition:
1. Add 60ul of fresh STET-E1 solution to each well in the plate, using the Multi-Drop.

2. Seal with Beckman foil seal using roller (when rolling the outside edges of the seal, press firmly to ensure a tight seal).

3. Vortex for 30 seconds with the VWR plate vortexer on low setting and immediately transfer to the incubators on metal shelves.

Cell Lysis
1. Incubate at 37C for 15 minutes

2. Incubate at 90C for 10 minutes

3. Incubate at -80 for 30 minutes if proceeding to fresh lysate step (If storing, freeze at -80OC).

Precipitation of cellular debris/plasmid DNA transfer
1. Centrifuge at 4000 rpm for 90 minutes.
(There is no need to thaw plates that have been stored at -80C before precipitating in the centrifuge).

2. Transfer 100ul of supernatant into a 96 well microtiter plate using the Hydra file #9. NOTE: the Hydra must aspirate only the top 100 ul, and must NOT aspirate the pellet. If more than 8 pellets are transferred, re-centrifuged liquid and pellet and reset the Hydra to a higher level.

3. Wash Hydra once with 2% Bleach and twice with DI H2O between transfers. At this point, the samples may be stored

4. Store transferred samples at - 20C overnight if needed.

Purification of plasmid DNA by SPRI
Binding, washing, drying and resuspension to the end of purifying plasmid DNA is completed using an automated DNA purification platform centered around a CRS articulated robotic arm. To set up the robot for SPRI purification, complete the following steps.

1. Add 60ml of prewashed magnetic beads to 500ml of PEG/NaCl solution, pour the mixture in to a 500ml Kimax beaker, and place the beaker into the agitator on the CRS platform.

2. Turn on the agitator and bring to a speed of 600rpm to prevent beads from settling to the bottom of the beaker.

3. Refill the plate washer reagent container with 70% ethanol.

4. Make sure plate washer waste container is empty.

5. Load the plate nest carousel with plates (without lids) of transferred supernatant (product of step 4). (Load only 70 plates at one time to prevent significant evaporation from the lidless plates.)

6. Make sure that each plate is completely within the metal positioning guides on each plate nest.

7. Home the CRS robotic arm and initialize the robot.

8. Press play!

In the case of CRS robot malfunction, it is necessary to complete the protocol without the aid of automation. The next 3 steps below explain how to complete the SPRI isolation protocol manually.

Binding of plasmid DNA:
1. Add 15ul of prewashed magnetic beads to each well using matrix pipettor.

2. Add 130ul of PEG solution using mutidrop.
(130ul of PEG is equal to a setting of 155ul on a Titertek Multidrop.)

3. Shake, uncovered, on medium speed (6-8) on plate shaker for 3 min., then let sit on benchtop for another 10 min. (If beads stick to sides of wells, use pipette tip to dislodge.)

4. Place on magnet plates for 10-15 min., or until the beads have aggregated into the bottom of the plate in a ring and the solution has cleared.

Washing:
Place microtiter plate on Biotek plate washer (SPRI Wash program) with 70% ETOH wash solution. SPRI Wash program includes 6 washes.

Drying, resuspension and elution:
1. Air dry on benchtop for 20 minutes.

2. Resuspend by adding 50ul of Millipore H2O using a Multidrop and shaking on medium (6-8) on plate shaker for 5-10 min.

Isolations QC- See Template Isolations QC SOP

DNA Heating
1. Check Oven temperature (Must be at 95C)

2. Place DNA SPRI Plate into 95C Oven for 35 min (Stack Plates 2 high if necessary)

3. Store at -20C

Reagent/Stock Preparation

TB STET-E1 (stable approximately 2 mo at room temp):
1M Tris pH 8.0, 10 mM EDTA, 1M NaCl, 4% tween-20
(STET solution is premade by Teknova)

For 230 Plates add following:(must be fresh daily)

820 mls	5M NaCl
230 mls	1M Tris/HCl pH 8.0
120 mls	500mM EDTA pH 8.0
230 mls	Tween 20

Add stir bar and stir 5 min at medium setting

Working STET lysis(solution must be fresh daily):
2 ml of 10 mg/ml RNAse stock solution, 2 ml of 50mg/ml Lysozyme to 100ml of STET-E1

*RNAse A stock solution:Dilute 10mg of Sigma RNAse A to 1ml with DI H2O.
*Lysozyme stock solution:Dilute 50mg of Lysozyme to 1 ml with DI H2O.

Magnetic bead suspension (stable approximately 2 mo at room temp):
*working solution:
1. prewashed magnetic beads diluted 1:10 in 10 mM Tris pH 8.0

2. Wash beads with 10 mM Tris: make an approximate 10% solution by measuring 5ml of beads in to a 50 ml conical tube with screw lid, add 10 mM Tris up tp 50ml mark, invert (do not shake, beads are fragile) several times to mix. Place tube on magnet holder, allow to settle for 5 minutes, and pour off liquid, repeat X3. Make a final solution 1:10 solution with the 10 mM Tris.

20% PEG- 2.5 M NaCl (Teknova) (double stranded hybridization solution).

Ethanol wash solution
70% ETOH (This wash should be made of fresh every day.)