Capillary-Based Sequencing Sample Prep

Detailed protocols on template preparation, PCR reaction mix preparation, cycling conditions, and post-PCR purification are available at http://www.appliedbiosystems.com. The protocols are summarized below.

Template Preparation

We recommend the Qiagen miniprep kit for isolating plasmid DNA and the Qiagen PCR purification kit to prepare PCR products.

Setting up Sequencing PCR reaction

The standard reaction can be halved or quartered to economize the BigDye Mix.

	Full	Half	Quarter
BigDye Reaction Mix	8.0 µL	4.0 µL	2.0 µL
Sequencing Primer	3.2 pmoles	1.6 pmoles	0.8 pmoles
DNA Template	15-30 fmoles	7-15 fmoles	3.5-7 fmoles
5X Sequencing Buffer*	0 µL	0 µL	1.0 µL
Total Reaction Volume	20 µL	10 µL	10 µL

*BigDye Reaction Mix contains 2.5X Sequencing Buffer. This must be supplemented with 5X Sequencing Buffer in quarter volume reactions.

PCR Cycle Sequencing

25 cycles of
96°C 10 seconds
50°C 5 seconds
60°C 4 minutes
hold at 4°C or 10°C

store at -20°C until ready to purify the reaction samples.

An MJ Research (Biorad) Tetrad thermocylcer programmed with these cycling conditions is available in the Bauer Core. It is saved in the Main menu and called “BIGDYE”.

Sample Purification

We strongly recommend column purification of the PCR sequencing samples. We most commonly use Centrisep columns from Princeton Separation, but similar products work as well. Samples may be resuspended in water or formamide at a minimum volume of 10uL. Samples must be submitted in 8-strip tubes (0.2uL tubes) or plates.

Core Reagents:

Following is a list of reagents mentioned in this document.

Item	Vendor	Part #
BigDye Terminator v 3.1 kit, (100 reactions)	Applied Biosystems	4337455
5X Sequencing Buffer (11mL)	Applied Biosystems	4305603
MicroAmp® 8-Tube Strips (125 strips)	Applied Biosystems	N8010580
Optical Caps, 8 Caps/Strip (300 strips)	Applied Biosystems	4323032
96 Well Plate	VWR	83009-676
Centrisep Spin Columns	Princeton Separations	CS-900/CS-901
Centrisep Spin Sep-8	Princeton Separations	CS-912

The Sequencing Facility sells 100-reaction aliquots of BigDye Terminator Reaction Mix. To purchase an aliquot, please fill out this form and email it to 3100user@mcb.harvard.edu. You can pick up your aliquot from the sequencing staff in NWL room B239. 1mL aliquots of 5X Sequencing Buffer are available free of charge to BigDye customers.