5-µL 384-Well Cycle Sequencing

Version Number:	1
Development Date:	3/28/00
Start RND date:	X
Author:	Damian Scott
Reviewed by:	Paul Richardson

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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
Microseal 'A' film	MJ	MSA-5001
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) 3) Using the Hydra, aliquot 3 ul of SPRI DNA on a magnetic plate into each well of a MJ V2.0 384 well plate on a Robbins plate adapter

4) 4) Using the Tecan, aliquot 2ul of ET cocktail into each well of the 384 well MJ V2.0 plate

5) Centrifuge the 384 well Robbins plate up to 1000rpm and back down in eppendorf centrifuge

6) 7) Place 384 well spun down MJ V2.0 plate into MJ 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. Visually check the bottom of the plate for well to well uniformity and note uniformity

4. Add 13ul/well of LiCl solution to the thermocycled 384 well plate

5. Immediately cover plate with clear seal

6. Place plate and holder into a VWR plate vortexer

7. Vortex 30 seconds (high setting)

8. Place 384 well plate into an Eppendorf 5810 centrifuge and close the lid

9. Centrifuge at 4000rpm for 90 minutes

10. After 90 minutes return to the centrifuge and immediately remove the plates (This must be done within 5 minutes after the rotor stops spinning)

11. 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)

12. Centrifuge at 350rpm for 1 minute

13. Carefully remove plates from centrifuge

14. 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)

15. Record formamide lot number and operator name

16. Add 10ul/well of formamide to air dried 384 well plate

17. Immediately cover plate with clear seal

18. Visual check plate for well to well uniformity

19. Centrifuge up to 1000rpm and back down

20. Vortex for 1 minutes in a VWR plate vortexer (maximum setting)

21. Place formamide plate in Eppendorf 5810 centrifuge

22. Centifuge at 1000rpm for 1 minute

23. Store plates in -20 C freezer

Reagent/Stock Preparation

ET Cocktail Preparation

1. Unfreeze ET kit on ice

2. Vortex 250pmol/ul working stock primer solution for 15 seconds using the vortex genie on high setting

3. Add 4ul of vortexed 250pmol/ul primer per 1ml of ET kit (keep on ice and covered from light)

4. Mix by inverting several times

LiCl Precipitation Preparation

1. Stir 1M LiCl for 2 minutes with a magnetic stir bar

2. Using a fresh bottle of 100% ETOH, add 180 ml of 100% and 3.6ml of 1M LiCl to a beaker

3. Place magnetic stir bar in beaker and mix on magnetic stirrer on medium setting for 2 minutes

Thermocycler conditions

Lid tracking +/- 1 C
Program: ETMB
95 C 25 sec
50 C 10 X 30cycles
60 C 2minutes
4 C HOLD

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Questions or Comments: Scott50@llnl.gov