Materials & Reagents

Materials/Reagents/Equipment	Vendor	Stock Number
Disposables
Purified DNA SPRI Plate	PSF/Isolations	From Library Creation
96 well Cycle Plate	Robbins Scientific	1055-90-0
CycleSeal PCR Plate Sealer	Robbins Scientific	1044-39-4
Microseal ?P? Sealing Pad	MJ Research	MSP-1001
Clear Plate Sealers	Edge BioSystems	48461
50 mL Centrifuge Tube	Falcon	2533-50
250 uL Pipet Tips	Rainin	RTL-250S
1000uL Pipet Tips	Rainin	RT-2205
Reagent Reservoir 100 ml	Matrix	8086
Stock Solutions
ET Terminator Mix	JGI	See Below
ETOH/Am-Ac Precipitation Mix	JGI	See Below
pUC 19 Standard Mix	JGI	See Below
Reagents
ET Terminator Kit - ET Terminator Premix - Formamide Loading Buffer	Amersham	US81095
Ammonium Acetate (7.5M solution)	Sigma	A2706
pUC 19 DNA Standard(1000 ng/ul)	NEB	304-1L
Primer - Forward -40M13 (250pmol/uL) GTT TTC CCA GTC ACG ACG TTG TA - Reverse -28M13 (250pmol/uL) AGG AAA CAG CTA TGA CCA T	Amersham	US81095
Ethanol (100%)	AAPER	N/A
Water (100%)	Millipore Milli-Q system	N/A
Equipment
MJ Thermocycler	MJ Research
PE 9700 Thermocycler	Perkin Elmer
96 Syringe Hydra (110uL)	Robbins Scientific
Multidrop 96 well	Titertek
96 well Ring Magnetic Plate	JGI
P20 Pipet	Rainin
P200 Pipet	Rainin
P1000 Pipet	Rainin
Plate Vortexer	VWR
Eppendorf 5810 Centrifuge	Eppendorf
Eppendorf 5416 Centrifuge	Eppendorf
Stir Bar	VWR
2L Glass Bottle	VWR
Hydra water wash trough	Robbins Scientific
Custom Magnetic Cart	JGI
96 well Plate Holder	Perkin Elmer

Procedure

Preparation of DNA Plates
1. Remove SPRI Isolated DNA plates from Sequencing Freezer #1 and place plates on magnets in custom magnetic cart
(If SPRI Isolated DNA plate is dropped and solution contacts Clear Plate Sealer discard plate and re-queue to template isolations)

2. Thaw SPRI Isolated DNA plates completely (1 to 3 hours)
(Plates may remain at room temperature for up to 6 hours)
Step 2 - shallow well plate thaw time is approx. 30 min., deep well approx 90 min

3. Make labels for the DNA Sequencing Plates on the web
Steps 3-4 - data entry is about 30 minutes per 50 plates

4. Discard water in 96 Syringe Hydra wash trough

5. Add 150 mls of fresh mill-Q water to each Hydra wash trough

6. Place Hydra wash trough on 96 Syringe Hydra stage and press wash button

7. Remove Hydra wash trough from 96 Syringe Hydra stage

8. Place SPRI Isolated DNA Plate on a separate 96 well Ring Magnetic Plate
(Leave until DNA solution is completely clear of brown beads ------ At least 30 seconds.)
Reject Plate if more than 5 wells are turbid with brown beads)

9. Carefully remove the Clear Plate Sealer from the SPRI Isolated DNA Plate

10. Place a SPRI Isolated DNA Plate with a 96 well Ring Magnetic Plate on each 96 Syringe DNA Hydra Stage
(Ensure that the plate is centered to avoid damage to the Hydra Syringes)

11. Press the Fill button on each 96 Syringe DNA Hydra

12. Remove the SPRI Isolated DNA Plate/96 well Ring Magnetic Plate assembly from each 96 Syringe DNA Hydra

13. Place the corresponding labeled Forward 96 well Cycle Plate with a 96 well Plate Holder on each 96 Syringe DNA Hydra

14. Press the Dispense button on each 96 Syringe Hydra

15. Remove the 96 well Cycle Plate and 96 well Plate Holder
(Stack plates to prevent evaporation and seal the top plate with a Clear Plate Sealer)
(Stack plates by primer direction)

16. Place the corresponding labeled Reverse 96 well Cycle Plate with a 96 well Plate Holder on each 96 Syringe DNA Hydra

17. Press the Dispense button on each 96 Syringe Hydra

18. Remove the 96 well Cycle Plate and 96 well Plate Holder
(Stack plates to prevent evaporation and seal the top plate with a Clear Plate Sealer)
(Stack plates by primer direction)

19. Place Hydra wash trough on 96 Syringe Hydra stage and press wash button

20. Remove Hydra wash trough on 96 Syringe Hydra stage
(Water in Wash Trough must be changed every ten plates) 21. Repeat steps 10 to 23 four times

22. Discard water in 96 Syringe DNA Hydra wash trough

23. Add 150 mls of fresh mill-Q water to each Hydra wash trough

24. Remove Hydra wash trough from 96 Syringe Hydra stage

25. Repeat steps 10 to 28 until batch is complete

Addition of PUC 19 Standard
1. Prepare pUC standard mix and place on ice

2. Place one 250 ul matrix tip on a 125 ul 8 channel matrix pipetor

3. Set matrix pipetor to fill (120 ul) , dispense 5 ul

4. Add 5 uL of the pUC 19 Standard mix into well A1 of each of your labeled sequencing plates
(Make sure to keep plates stacked to prevent evaporation)

Sequencing Premix Addition
1. Prepare Sequencing reagent premix

2. Invert sequencing reagent premix six times

3. Place sequencing reagent premix on ice
(Keep covered with aluminum foil and on ice at all times)

1. Reattach multidrop tube manifold to multidrop

2. Check multidrop calibration using calibration protocol #1

3. Place multidrop feed tubes into sequencing reagent premix solution

4. Seal top of bottle with parafilm.

5. Prime multidrop with sequencing reagent premix solution until liquid comes out of all eight multidrop nozzles.
(Ensure that the multidrop feed tubes contain no air bubbles)

6. Check multidrop settings (volume=5 ul, columns = 12)

7. Place an empty new 96 well Cycle Plate in the sequencing reagent premix multidrop

8. Press start key

9. Visually check the bottom of the plate for well to well uniformity
(If the multidrop fails either QC, contact your supervisor and recalibrate the instrument)

10. Insert DNA plate into multidrop

11. Check to ensure that multidrop feed tubes are immersed in the sequencing reagent premix solution

12. Press start key

13. Immediately cover plate with clearseal

14. Visual check plate for well to well uniformity
(If more than 10 wells have varying volumes, reject and contact your supervisor)

15. Stack sets of 2 into each plate holder of the Eppendorf 5416 Centrifuge

16. Place into an Eppendorf 5416 centrifuge and close the lid

17. Lock lid and spin 600 rpm for 1 minute

18. Place plates on ice and transport to thermal cycler room

19. Place plate in thermocycler (MJ Research Tetrad or PE 9700)

20. On MJ tetrad --- Turn blue thumbwheel counterclockwise 3 turns

21. Latch lid closed

22. On MJ tetrad - Turn blue thumbwheel clockwise until lid contacts plate and gives a slight increase in resistance

23. Turn blue thumbwheel ? turn clockwise for proper pressure

24. Select the ETMB program for each plate

25. After 15 min, check each thermocycler to ensure is running properly

26. If thermocycler is not running --- transfer the sequencing plate to another thermocycler

Post Sequencing Reaction Cleanup/Precipitation
1. Prepare Ammonium Acetate/Ethanol solution(Am-Ac/EtOH)

2. Retrieve sequencing plates from thermocyclers
(Plates must be covered and placed on ice)

3. Reattach multidrop tube manifold to multidrop

4. Check multidrop calibration using multidrop calibration protocol #1

5. Place multidrop feed tubes into Am-Ac/ETOH solution

6. Seal top of bottle with parafilm.

7. Prime multidrop with 10 mL of Am-Ac/ETOH solution.
(Ensure that the multidrop feed tubes contain no air bubbles)

8. If the multidrop lines contain air bubbles continue to prime until they are removed

9. Check multidrop settings (volume=100 ul, columns = 12)

10. Visually check the bottom of the plate for well to well uniformity
(If the multidrop fails either QC, contact your supervisor and recalibrate the instrument)

11. Insert sequenced plate into multidrop

12. Check to ensure that multidrop feedtubes are immersed in the Am-Ac EtOH solution

13. Press start key

14. Immediately cover plate with clearseal

15. Visual check plate for well to well uniformity
(If more than 10 wells have varying volumes, reject and contact your supervisor)

16. Transfer the plate to a 96 well Plate Holder

17. Place plate and holder into a VWR plate vortexer

18. Stack in 4 sets of 2 plates with holders

19. Adjust top of vortexer to firmly hold plates in place

20. Vortex 2 minutes (setting = 6)

21. Stack sets of 2 into each plate holder of the Eppendorf 5810 Centrifuge
(Place a rubber pad between each stack to prevent plate damage)

22. Place into an Eppendorf 5810 centrifuge and close the lid

23. Select program 1 and check centrifuge settings
(speed = 4000 rpm, time = 90 minutes)

24. Press start

25. In 15 minutes check the centrifuge to ensure it is running properly

26. After 89 minutes return to the centrifuge and immediately remove the plates
(This must be done within 5 minutes after the rotor stops spinning)

27. Carefully invert the plate and pour the Am-Ac/EtOH solution into the waste container
(Ensure that the pellet is not dislodged from the bottom of the plate) 28. Place a paper towel on top of the plate, invert and place in Eppendorf 5810 centrifuge

29. Select program 2 and check centrifuge settings
(speed = 600 rpm, time = 1 minute)

30. Press start

31. Carefully remove plates from centrifuge

32. Place sample plates into drawers marked air dry for 15 minutes
(Plates must be diluted with formamide within 30 minutes or placed in the -20 C freezer)

33. Retrieve formamide loading buffer solution from sequencing chemistry freezer 2

34. Record formamide lot number and operator name

35. Reattach multidrop tube manifold to formamide multidrop #1

36. Check multidrop accuracy with multidrop calibration protocol #1

37. Place multidrop feed tubes into formamide loading solution

38. Seal top of bottle with parafilm.

39. Prime multidrop with 10 mL of formamide loading solution.
(Ensure that the multidrop feed tubes contain no air bubbles)

40. If the multidrop lines contain air bubbles continue to prime until they are removed

41. Check formamide multidrop settings (volume=10 ul, columns = 12)

42. Place an empty new 96 well Cycle Plate in the formamide multidrop

43. Press start key

44. Immediately cover plate with clear seal

45. Visual check plate for well to well uniformity
(If more than 10 wells have varying heights reject and contact your supervisor)

46. Place formamide plate in a 96 well Plate Holder

47. Place plate and holder into a VWR plate vortexer

48. Stack in 4 sets of 2 plates with holders

49. Vortex for 2 minutes in a VWR plate vortexer
(setting = 6 )

50. Place formamide plate in Eppendorf 5810 centrifuge

51. Select program 2 and check centrifuge settings
(speed = 600 rpm, time = 1 minute)

52. Press start

53. Carefully remove plates from centrifuge upon completion of spin

54. Transfer plates to Electrophoresis Freezer #1

Reagent/Stock Preparation

pUC 19 Standard Mix
1. Retrieve a tube of pUC 19 standard (1000ng/ul) from sequencing chemistry freezer #2

2. Place on ice and allow to thaw
(usually one hour)

3. Record pUC19 lot # and operator name

4. Retrieve two new 1.5ml Eppendorf centrifuge tubes

5. Mark one tube pUC

6. Turn on the Milli-Q water system and let it run for 20 seconds

7. Fill one Eppendorf tube with Milli-Q water

8. Using a pUC P1000 pipetor, transfer 950 uL of Milli-Q water to the pUC labeled tube

9. Vortex pUC19 standard for 5 seconds with tube vortexer
(setting = 5)

10. Using a pUC P200 pipetor, transfer 50 uL of the pUC 19 to the pUC labeled tube

11. Vortex the pUC labeled tube for 5 seconds with tube vortexer
(setting = 5)

12. Place pUC tube on ice
(use within 4 hours and discard)

Sequencing Premix Solution
1. Retrieve ET terminator stock mix from sequencing chemistry freezer #3

2. Place ET stock mix on ice and cover with foil

3. Thaw for 1 hour

4. Record lot number and operator name

5. Retrieve Forward and Reverse Primer(250uM/ul) from Sequencing Chemistry Freezer #2

6. Place Primer tubes on ice and thaw for 1 hour

7. Invert ET Terminator stock mix 6 times.
(avoid harsh mixing because the solution is unstable)

8. Retrieve a 50ml falcon tube and rinse twice with Milli-Q water

9. Cover falcon tube with aluminum foil and place on ice

10. Add the following to a 50 mL falcon tube covered in foil:

#96 Plates to Sequence	1 Plate	50 Plates	100 Plates
ET Terminator Mix	400 uL	20 mL	40 mL
Milli-Q H2O	100 uL	5 mL	10 mL
Primer	2 uL	100 uL	200 uL

11. Invert covered falcon tube 6 times and store on ice.

Am-Ac/ETOH Precipitation Preparation (300 plates) 1. Retrieve a 2 liter glass bottle, 1 liter ethanol graduated cylinder and a 100 ml Am-Ac graduated cylinder

2. Wash each cylinder and bottle with 20 mls of 100% Ethanol
(Ensure all the ethanol is removed)

3. Retrieve 2 bottles of fresh 100% Ethanol from the flammable cabinet in room number

4. Retrieve 1 bottle of 7.5M ammonium acetate from sequencing chemistry freezer #2

5. Measure 826 mL 100% Ethanol in the ethanol graduated cylinder

6. Cover with parafilm or transfer immediately to 2 liter glass bottle

7. Measure 172.5 mL Milli-Q H2O in the ethanol graduated cylinder

8. Transfer to 2 liter glass bottle and seal bottle top

9. Measure 27.5 mL of Ammonium Acetate in the Am-Ac graduated cylinder

10. Transfer to 2 liter glass bottle and seal bottle top

11. Add a clean stir bar and place on VWR stir plate

12. Stir on medium for 10 minutes
(Make fresh everyday and discard in proper waste container at the end of the day)

Instrument Settings

MJ Research Tetrads
Program ETMB
950C(25sec)
500C (10sec)
600C (2min)
30 cycles
Hold at 40C
Lid +150C tracking
Plate
Sample temperature = calculated

Perkin Elmer 9700
Program ETMB
950C(25sec)
500C (10sec)
600C (2min)
30 cycles
Hold at 40C

Multidrops
Formamide Multidrop
Column = 12 Volume = 10

Am-Ac EtOH Multidrop
Column = 12 Volume = 100

Hydra
DNA Transfer Hydra?s
File = 1
Mode = Dis
DV = 5.0
DH = 3800
FV = 12.0
FH = 3740
EH = 3740
WV = 20.0
WH = 3600
Wash = 3
TD = 0

Chemical Storage

Sequencing Chemistry Freezer #1
Location = Room 121
Temperature = -200C
Contents: Submitted Purified DNA SPRI Plate

Sequencing Chemistry Freezer #2
Location = Room 121
Temperature = -200C
Contents:
7.5 M Ammonium Acetate (50 ml)
Formamide Loading Buffer (100 ml)

Sequencing Chemistry Freezer #3
Location = Room 121
Temperature = -200C
Contents: Sequenced SPRI Template DNA Plates

Sequencing Chemistry Freezer #4
Location = Room 121
Temperature = -200C
Contents:
ET Terminator Mix Stock
Diluted ET Terminator Sequencing Mix with Primer
Primer Forward ?40M13 (250pmol/uL)
Primer Reverse ?28M13 (250pmol/uL)

Flammable Locker
Location = Room 121
Temperature = Room Temperature
Contents: 100% Ethanol