MegaBACE ET Terminator Protocol (96 well) |
Version Number: | 3 |
Start Production Date: | 11/01/99 |
Author: | Susan Lucas |
Edited by: | Chris Elkin |
Reviewed by: | Paul Predki, Jamie Jett |
Summary |
Materials & Reagents |
Materials/Reagents/Equipment | Vendor | Stock Number |
---|---|---|
Disposables | ||
Purified DNA SPRI Plate | PSF/Isolations | By Library |
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 | GIBCO_Life Technologies | 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 | |
1L Graduated cylinder | 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 Refrigerator #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 10 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 8 to 20 nine 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 8 to 23 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 (250 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)
4. Reattach multidrop tube manifold to multidrop
5. Check multidrop calibration using calibration protocol #1
6. 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)
7. Place multidrop feed tubes into sequencing reagent premix solution
8. 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)
9. Check multidrop settings (volume=5 ul, columns = 12)
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. Visual check plate for well to well uniformity
(If more than 10 wells have varying volumes, reject and contact your supervisor)
14. Stack sets of 4 into each plate holder of the Eppendorf 5416 Centrifuge
15. Place into an Eppendorf 5416 centrifuge and close the lid
16. Lock lid and spin 600 rpm for 10 seconds
17. Place plates on ice and transport to thermal cycler room
18. Place plate in thermocycler (MJ Research Tetrad or PE 9700)
19. On MJ tetrad --- Turn blue thumbwheel counterclockwise 3 turns
20. Latch lid closed
21. On MJ tetrad - Turn blue thumbwheel clockwise until lid contacts plate and gives a slight increase in resistance
22. Turn blue thumbwheel ? turn clockwise for proper pressure
23. Select the ETMB program for each plate
24. After 15 min, check each thermocycler to ensure is running properly
25. 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)
Post Sequencing Reaction Formamide Addition
1. Retrieve formamide loading buffer solution from sequencing chemistry freezer 2
2. Reattach multidrop tube manifold to formamide multidrop #1
3. Check multidrop accuracy with multidrop calibration protocol #1
(If the multidrop fails either QC, contact your supervisor and recalibrate the instrument)
4. Place multidrop feed tubes into formamide loading solution
5. Seal top of bottle with parafilm.
6. Prime multidrop with 10 mL of formamide loading solution.
(Ensure that the multidrop feed tubes contain no air bubbles)
7. If the multidrop lines contain air bubbles continue to prime until they are removed
8. Check formamide multidrop settings (volume=10 ul, columns = 12)
9. Place an empty new 96 well Cycle Plate in the formamide multidrop
10. Press start key
11. Immediately cover plate with clear seal
12. Visual check plate for well to well uniformity
(If more than 10 wells have varying heights reject and contact your supervisor)
13. Place formamide plate in a 96 well Plate Holder
14. Place plate and holder into a VWR plate vortexer
15. Stack in 4 sets of 2 plates with holders
16. Vortex for 2 minutes in a VWR plate vortexer
(setting = 6 )
17. 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 |
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
95 C(25sec)
50 C (10sec)
60 C (2min)
30 cycles
Hold at 4 C
Lid +15 C tracking
Plate
Sample temperature = calculated
Perkin Elmer 9700
Program ETMB
95 C(25sec)
50 C (10sec)
60 C (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 139
Temperature = -4 C
Contents: Submitted Purified DNA SPRI Plate
Sequencing Chemistry Freezer #2
Location = Room 140
Temperature = -20 C
Contents:
7.5 M Ammonium Acetate (50 ml)
Formamide Loading Buffer (100 ml)
ET Terminator Mix Stock
Diluted ET Terminator Sequencing Mix with Primer
Primer Forward -40M13 (250pmol/uL)
Primer Reverse -28M13 (250pmol/uL)
Sequencing Chemistry Freezer #3
Location = Room 141
Temperature = -20C
Contents: Sequenced SPRI Template DNA Plates
Sequencing Chemistry Freezer #4
Location = Room 141
Temperature = -20 C
Contents: Sequenced SPRI Template DNA Plates
Flammable Locker
Location = Room 121
Temperature = Room Temperature
Contents:
100% Ethanol