384-Well Cycle Sequencing
| Version Number: | 1 |
| Development Date: | 3/28/00 |
| Start RND date: | X |
| Author: | Damian Scott |
| Reviewed by: | Paul Richardson |
Summary
384-well cycle sequencing using ET Terminator kit and Lithium Chloride/Ethanol solution for precipitation.
Materials & Reagents
| Materials/Reagents/Equipment | Vendor | Stock Number |
|---|---|---|
| Disposables | ||
| Collection microtubes | Qiagen | 19560 |
| 384 well plates | Perkin Elmer | 4305505 |
| 384 well plates Version 2 | MJ | MSP-3852 |
| Clear Plate Sealers | Edge BioSystems | 48461 |
| Stock Solutions | ||
| 250pmol/ul primers | IDT | N/A |
| LiCl/Etoh Precipitation Mix | JGI-SEE Below | N/A |
| Formamide Loading Buffer | Amersham | US81095 |
| Reagents | ||
| ET Kit | Amersham | US81090 |
| 8M LiCl solution | Sigma | L7026 |
| Ethanol (100%) | AAPER | N/A |
| Water (100%) | Millipore Milli-Q system | N/A |
| Equipment | ||
| Tecan | Tecan | N/A |
| Hydra 96 | Robbins | N/A |
| PE 9700 Thermocycler | Perkin Elmer | N/A |
| MJ thermocycler | MJ | N/A |
| Robbins Plate adapter | Robbins | N/A |
| Eppendorf 5810 Centrifuge | Eppendorf | N/A |
| Eppendorf 5416 Centrifuge | Eppendorf | N/A |
| Stir Bar | VWR | N/A |
| 2L Glass Bottle | VWR | N/A |
| Plate Vortexer | VWR | N/A |
Procedure
Cycle Sequence
1) Unfreeze the ET kit, primers, and SPRI DNA, prepare solutions, and place everything on ice
2) Place unfrozen SPRI DNA plate(s) on magnetic plate(s)
3) Using the Hydra, aliquot 5 ul of SPRI DNA on a magnetic plate into each well of a PE 384 well plate on a Robbins plate adapter
4) Using the Tecan, aliquot 5ul of ET cocktail into each well of the 384 well Robbins plate
5) Centrifuge the 384 well Robbins plate up to 1000rpm and back down in eppendorf centrifuge
6) Place 384 well spun down Robbins plate into PE thermocycler using the ETMB program
7) Precipitate using the LiCl precipitation protocol
Post Sequencing Reaction Cleanup/Precipitation
1. Prepare Lithium Chloride solution(LiCl)
2. Retrieve sequencing plates from thermocyclers
3. Add 28.5ul/well of LiCl solution to the thermocycled 384 well plate
4. Visually check the bottom of the plate for well to well uniformity
5. Immediately cover plate with clear seal
6. Place plate and holder into a VWR plate vortexer
7. Adjust top of vortexer to firmly hold plates in place
8. Vortex 30 seconds (medium setting)
9. Place 384 well plate into an Eppendorf 5810 centrifuge and close the lid
10. Centrifuge at 4000rpm for 90 minutes
11. After 90 minutes return to the centrifuge and immediately remove the plates (This must be done within 5 minutes after the rotor stops spinning)
12. Carefully place a paper towel on top of the plate, invert and place in Eppendorf 5810 centrifuge (Ensure that the pellet is not dislodged from the bottom of the plate)
13. Centrifuge at 350rpm for 1 minute
14. Carefully remove plates from centrifuge
15. 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)
16. Record formamide lot number and operator name
17. Add 10ul/well of formamide to air dried 384 well plate
18. Immediately cover plate with clear seal
19. Visual check plate for well to well uniformity
20. Place plate and holder into a VWR plate vortexer
21. Vortex for 1 minutes in a VWR plate vortexer (maximum setting)
22. Place formamide plate in Eppendorf 5810 centrifuge
23. Centifuge at 1000rpm for 1 minute
24. Press start and when the centrifuge reaches 1000rpm press stop.
25. Carefully remove plates from centrifuge upon completion of spin
26. Store plates in -20 C freezer
Reagent/Stock Preparation
ET Cocktail Preparation
1)Dilute unfrozen ET kit on ice (1 part Milli-Q water:4parts ET kit)
2)Vortex 250pmol/ul working stock primer solution for 10 seconds using the vortex genie on high setting
3)Add 4ul of vortexed 250pmol/ul primer per 1ml of ET kit (keep on ice)
4)Mix by inverting several times
5)Aliquot ET cocktail into 8 Qiagen collection microtubes (keep on ice and covered from light until use)
LiCl Precipitation Preparation
1. Retrieve a 2 liter glass bottle, 1 liter ethanol graduated cylinder and a 100 ml LiCl 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
4. Retrieve 1 bottle of 1M LiCl
5. Measure 1500ml 100% Ethanol in the ethanol graduated cylinder
6. Transfer immediately to 2 liter glass bottle
9. Measure 30 mL of Lithium Chloride in the LiCl 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 2 minutes
Thermocycler conditions
Program: ETMB
95 C 25 sec
50 C 10 X 30cycles
60 C 2minutes
4 C HOLD
Questions or Comments: Scott50@llnl.gov
