DNA Sequencing - Reaction Parameters

As stated in our chemistry section, template purity is vital for achieving the best possible result. Additionally, severely contaminated template DNA can permanently affect the linings of our sequencer capillaries resulting in a loss of basecall resolution.

We require that ALL template DNA to be used in the cycle sequencing reactions submitted to our facility, be post-extraction purified to eliminate the contaminants that cause capillary degradation and reaction failure.

PLEASE NOTE:
Our requirement is that you use one of the many standard, commercially available column-based, DNA purification kits that are appropriate for the type of template you are using. We can provide a list of commonly-used products and suppliers if needed. The only non column-based or chemical method that is acceptable at Micromon is the AB modified alkaline lysis-PEG precipitation method.

The operator's manual for the Applied Biosystems BigDye Terminator Cycle Sequencing Ready Reaction Kit V3.1 is a useful guide for background information and the set up of the cycle sequencing reactions. Micromon has developed its own modifed Reaction Set Up Protocol which is tailored for our 3730 DNA sequencer and is the recommended protocol for users of our service. Together with our Reaction Clean Up Protocol to remove the residual unincorporated BigDye terminators, these methods comprise the fundamental procedures of the cycle sequencing process.

In addition to high template purity, good primer design is a significant factor in successful DNA sequencing.

BigDye Reaction Buffer
Customers who purchase the BigDye v3.1 mix from us, can also be supplied with the Applied Biosystems 5x reaction buffer. Regrettably, we are not given sufficient buffer by AB to cover the standard set up of a reaction for processing on our 3730 analyzer. Therefore customers have several options:

a) purchase additional buffer from Applied Biosystems (this is an extremely expensive option)

b) use the buffer supplied with any brand of Taq DNA Polymerase that contains MgCl2 (usually supplied as a 10x stock)

c) we can supply free of charge, a 10x stock of Roche Taq PCR buffer - in trials here at Micromon, this buffer produces results no different to the AB buffer

d) make up your own 5x buffer stock using the AB recipe [400mM Tris pH 9.0, 10mM MgCl2].

Despatching Orders to Micromon

Please send your dried samples in well-labelled, 1.5 ml microfuge tubes, together with a completed order form. Orders can be delivered in person, by courier or by post.

We strongly recommend that customers use filenames in the format [your initials][consecutive numbers between 1-1000] otherwise please keep filenames to a maximum 10 characters.