Materials & Reagents

Materials/Reagents/Equipment	Vendor	Stock Number
Disposables
Latex Exam Gloves	N/A	N/A
Weigh boat	N/A	N/A
Reagent Reservoirs, 25 ml	Matrix	N/A
Micro Centrifuge tubes,1.5 ml	Applied Scientific	N/A
Micro Centrifuge tubes,0.5 ml	Eppendorf	N/A
Aluminum Foil Lids	Beckman	N/A
Kim Towels	Kimberly Clark	N/A
Reagents
10X TBE	Teknova	N/A
Agarose	Gibco BRL	N/A
Ethidium Bromide	Gibco BRL	N/A
STOP Solution	JGI	N/A
Plasmid pUC 18 DNA (.25 ug/ul)	Gibco BRL	N/A
Equipment
Spatula	N/A	N/A
96 well .2 ml thin wall PCR plates	Applied Scientific	N/A
PCR Plate Sealers	Robbins Scientific	N/A
Centrifuge 5416	Eppendorf	N/A
Mini Centrifuge	VWR	N/A
P-20, P200, P-1000 Pipetmen	Gilson	N/A
.5-10 ul Finnpipette	Applied Scientific	N/A
125 ul 8-Channel Pipettor	Matrix	N/A
Yellow tips	N/A	N/A
1250 ul Pipet tips	Matrix	N/A
.1-10 ul pipet tips	Applied Scientific	N/A
Delta Range Scale	Mettler	N/A
Agar Sterilizer AS-10	New Brunswick Scientific	N/A
505 Dz Pump	Watson Marlow	N/A
Tecan Genesis RSP 200	Tecan	N/A
Gel Casting System 20-25	Gibco BRL	N/A
Gel transport racks	LBL	N/A
Gel Electrophoresis System	LBL	N/A
Fluor MultiImager	BioRad	N/A
Forced Air Oven	VWR	N/A
Isotemp Oven	Fisher Scientific	N/A

Procedure

Gel Casting Procedure:

Prepare 1.4% Agarose gel solution:
1. In the Agar Sterilizer dilute 10X TBE with ddH20 to make the desired volume of 1X TBE.

2. Weigh out and add appropriate amount of agarose for the desired volume.

3. Screw the top on tightly and reconnect all fittings.

4. Begin the sterilization program.

5. When gel solution has reached the Dispensing temperature of 65C (approximately after 1 1/2 hours), add appropriate volume of Ethidium Bromide and mix.

6. Always record on tank the date, operator�s initials, volume made, and whether or not EtBr has been added.

Cast the gels:
1. Put on latex gloves.

2. Set up gel trays on sled racks with clamps at the end and three combs with the ridged side facing the bottom of the gel.

3. Attach dispensing tube to the harvesting valve of the agar sterilizer.

4. Dispense 400 ml of agarose solution into each gel tray using the peristaltic pump.

5. Empty agarose remaining in the tube back into the agar sterilizer. Flush the tubing with warm water and flush the water out with air.

6. Record on tank the date, operator�s initials, and number of gels poured.

7. Allow the gels to set for 30 minutes before using.

Preparing samples in PCR plates:

Using the Tecan to prepare PCR plates:
1. Before using the Tecan, check the ddH2O and waste water levels in the two tanks to the left of the machine. Fill/empty as necessary.

2. Place PCR plates in black adaptors in the center of the Tecan�s loading platform.

3. Fill a 25 ml Reservoir with STOP solution and place in the marked area below the black plates on the loading platform.

4. Place SPRI plates on magnetic adaptors on the loading platform. A maximum of 24 can be loaded at once (12 SPRI plates max per PCR plate). Push SPRI plates and their magnets up and to the left within the adaptor for optimal positioning for the Tecan. Make sure the PCR plates are labeled to reflect the order of the SPRI plates on the loading platform.

5. Double click on the "Tecan Logic Software" icon.

6. At the beginning of the day, flush before using. Click on the "Flush" button and click "OK".

7. Click on the "Start" button. Select "Prepare X Plate(s) for Gel Loading". Select 1 or 2 depending on how many SPRI plates there are to load. Click "OK".

8. Enter the number of samples. Enter "8" for 1 PCR plate or "16" for 2 PCR plates. Click "Save".

9. Tecan will load 6 ul STOP solution per well and add an additional 6 ul template DNA from the SPRI plates for a total volume per well of 12 ul.

10. Seal finished PCR plates and store in 4 C deli fridge in the bin for that day.

Preparing PCR plates by hand:
1. Place SPRI plates on magnetic plate adaptors. Let stand for a few minutes to allow the magnetic beads to settle.

2. Using an 8-channel pipettor, aliquot 6 ul of STOP solution per well into a PCR plate.

3. Using an adjustable 8-channel Matrix pipettor, take 6 ul from the SPRI plate in a diagonal pattern starting with well A1. Empty the sample into the labeled PCR plate with STOP solution.

4. Seal PCR plates and store in 4 C deli fridge.

Additional Processing of SPRI plates following QC procedure:
1. Seal SPRI plates following preparation of the PCR plates with aluminum foil sealers.

2. Heat in oven for 35 min. at 95 C.

3. Store in -20 C freezer pending pass/fail by the gel pictures.

Gel Loading Procedure:

Preparing the Tecan to load gels:
1. Retrieve PCR plates containing STOP solution and template DNA from the deli fridge in Room 139.

2. Label plates with the gel number and plate number of that gel.

3. Place PCR plates into plate holders located on the Tecan and remove plate sealers.

4. Remove gels from the casting trays, submerge in 1X TBE in the electrophoresis boxes, drain off excess, and lay out on the loading platform. Label the gels with the appropriate gel number for the PCR plates laid out on the loading platform.

5. Place 3 sets of 3 plasmid pUC 18 concentration standards into the tube holders in the center of the deck in the following orientation:

20	80
	300

Loading Gels
1. Before using the Tecan, check the ddH2O and waste water levels in the two below the machine. Fill/empty as necessary.

2. Double click on the "Logic V.1.92" icon.

3. At the beginning of the day, flush before using. Click on the "Flush" button and click "OK".

4. Click on the green "Start" button.

5. Select one of the programs (based on the number of gels to be loaded) under "LOAD DNA QUANT GEL". Click "OK".

6. Tecan will load 10 ul of sample per well from the PCR plates on the gel and 10 ul of the concentration standards per well to give a total of 20, 80, and 300 ng of DNA in the designated wells.

Electrophoresis:

Prepare the gel boxes
1. Fill gel boxes with 1X TBE without EtBr.

2. Top off gel boxes with ddH2O.

Load the gels into the gel boxes
1. Make sure loaded gels are submerged in buffer.

2. Close the drawers completely.

3. Check that the voltage on the power supply is set at 250 V and the timer is set for 45 min.

4. Press green button to start run. "Run" should flash in the timer window and time should begin 45 min. countdown.

Imaging Gels:

Remove the gels from the electrophoresis boxes and drain away any excess buffer.

Using the MultiImager:
1. Double click on the MultiAnalyst icon to enter program.

2. Go to file and select "New".

3. Change Integration Time to 30 seconds and Light Source to EPI: UV.

4. Place the gel, still on its tray, onto the imaging surface, snug up against the metal adaptor. The top of the gel should be on the left.

5. Click Capture.

6. When the second window pops up, click on "Tr". Using the mouse, slide the black arrow on the right over to the left until it is at the right-hand edge of the shaded region. Adjust the exposure as necessary.

7. Click OK.

8. Go to File and select "Save As" and enter the file name "Month_Day_YeargelX".

9. Go to File and select "Print".

10. Remove the gel tray from the imager. Dispose of gel in the marked bin and rinse off tray with distilled H2O. Place tray in rack to dry.

11. Rinse off imaging surface with ddH2O and dry with Kim Towel.

Labeling Gel Pictures/Entering Gels into Log:

Labeling Gel Pictures
1. On the gel printout, mark off blocks of 8 lanes (remember to take the 3 sets of 3 lanes of concentration standards into account) that represent the samples from a given plate. 2. Label the regions using the PCR plates the samples were loaded from. Remember Column 1 from Plate 1 loaded into the top right wells, loading from right to left. Entering into the Log
1. Open the Isolations folder on the Desktop.

2. Open the Gel Logs folder.

3. Open the file "Gel Logs template"

4. Enter the date, gel number, operator�s initials, and plate names in the appropriate columns from the PCR plates. Enter the names in the same order that they were loaded in.

5. Go to File and select "Save As" and enter the date as the file name. Click "OK".

6. Drag the file to the Gel Logs folder on the KINGCOBRA server to copy it.

Place the most recent pictures into the back of the log book.

Disposal of Gels

1. After gels have air-dried in the bin to some extent, place them in the oven on aluminum foil to complete the drying process. Always wear gloves when handling the gels!

2. Dispose of dried gels in the trash.

Basic Maintenance

1. Agar Sterilizer: Check rubber rings and fittings, vents, and plastic parts on a regular basis. Replace as necessary. Keep surfaces and surrounding area clear.

2. Gel Casting: Check combs for missing teeth. Check levels of sled racks and adjust when necessary. Check silicone tubing for the pump.

3. Electrophoresis: Change the running buffer every 2 weeks. Clean boxes, lids, and shelves with warm water.

4. Tecans: Wipe down the decks. Flush on a regular basis. Check needles and calibrate as necessary.

5. MultiImager: Regularly wipe down imaging surface.

Reagent/Stock Preparation

1X TBE:
From 10X TBE:
10 L: 1 L 10X TBE + 9 L ddH2O
7.5 L: 750 ml 10X TBE + 6.75 L ddH2O

To Make 1.4% Agarose Solution in the Agar Sterilizer:
10 L: 1 L 10X TBE + 9 L ddH2O + 140 g Agarose
Wait until at Dispense Temperature (65 C) before adding 100 ul Ethidium Bromide.

Helpful Hints:
1.4% agarose solution = 1.4 g agarose per 100 ml 1X TBE or 14 g per liter.
1 ul Ethidium Bromide per 100 ml agarose solution or 10 ul per liter.

To Make 20, 80, and 300 ng Concentration Standards from Plasmid pUC 18 stock:
For 900 ul (store in 1.5 ml Micro Centrifuge tubes and aliquot into .5 ml tubes for loading with Tecan)
20 ng: 7.2 ul stock + 892.8 ul STOP solution
80 ng: 28.8 ul stock + 871.2 ul STOP solution
300 ng: 108 ul stock + 792 ul STOP solution
Invert to mix and spin down briefly before aliquot/storage in -20 C freezer.

To Make STOP solution:
In 1 L bottle add:
200 g sucrose
10 ml 1M EDTA, pH 8.0
50 ml .5% bromphenol blue
500 ml ddH2O
Add stir bar and stir until sucrose is dissolved. Filter before use.