dLUTE SEQ CF™ is a version of the dLUTE SEQ DNA sequencing dilution reagent optimized for small scale users and core facility users. This version is simpler and optimize to use and offers the following advantages:
- Sequencing reactions require only 0.2 µl of ABI BigDye DNA sequencing premix reagent. This equivalent to a 1:40 dilution of a standard reaction.
- Reaction substrates are maintained at the optimum concentration resulting in longer reads and more robust results when sequencing DNA.
- Supports a wider range of DNA template concentrations and quality.
- Avoids the need to purchase new high precision liquid handling equipment.
Figure 1. The two phase states of dLUTE SEQ CF. Left. Set-up state. The aqueous phase (pink) is dispersed throughout the inert phase. Right. Reaction state. The aqueous reaction-phase (red) has separated from the inert phase (clear).
Ultra sensitive DNA sequencers, such as the Applied Biosystems 3100 and 3730, and the Amersham Biosciences MegaBACE 1000 and 4000, require a small fraction of the labeled sequencing fragments created in a full scale DNA sequencing reaction. Consequently, many sequence centers have focused on reducing sequencing costs by scaling down the reaction volumes and/or by using large dilutions of the premix reagent. Using this approach many core facilities and small users have been able reduce the amount of DNA sequencing premix chemistry required to 1/8 to 1/16th of that required for full scale reactions.
While these approaches have yielded major cost savings, the use of higher reagent dilutions can have a negative impact on the trace read lengths and on the success rate due to the enzymatic sequencing reaction being to substrate limitated. While it is possible to avoid some of these problems by setting up smaller reaction sizes, evaporation in the PCR tube means that reactions less than four microlitres a unreliable. Because of these problems, sequencing reactions are currently performed at scales far in excess of the true sensitivity of modern DNA sequencing instruments. The dLUTE SEQ CF sequencing reagent avoids these problems by using a novel two state formulation chemistry (Figure 1). In the first state (setup state), the aqueous sequencing reagents are dispersed in a large volume of the inert component of the dLUTE formulation. This effectively dilutes the aqueous reagents and enables pipetting with standard equipment. The dLUTE SEQ CF DNA sequencing reaction is then induced by heat to enter the second (reaction) state. In this state, the aqueous phase separates from the inert phase forming a small “bubble” (in which the DNA sequencing reaction takes place) overlain by the inert phase. This insert phase prevents evaporation of the small reaction volume allowing small volumes reaction to be performed successfully.
Feature and benefits of dLUTE SEQ CF
Improved sequencing data
The dLUTE SEQ reagent maintains the sequencing DNA polymerase with the optimal nucleotide and dye labeled terminator concentrations. This avoids the “ski-slope” effect (i.e. rapid fall off in signal strength) observed with excessive chemistry dilutions.
Ease of implementation
The dLUTE SEQ CF into existing production pipelines is straightforward and demands only minor modification to existing protocols. The DNA sequencing chemistry and oligo primer are simply mixed with the dLUTE SEQ reagent to form a stable homogenous, mixture. This mix is aliquoted onto 1 µl of DNA sequencing template using standard equipment. The inert phase overlays the small reaction volume and prevents the small sample from evaporating. The dLUTE SEQ CF sequencing reaction can be cleaned up using any of a number of compatible methods including SPRI beads, size-exclusion spin columns or plates, or via a simple modified alcohol precipitation protocol. The formulation of dLUTE SEQ CF use the same dLUTE component as the standard dLUTE SEQ reagent but is supplied with a different phase indicator buffer and protocol. The dLUTE SEQ CF reagent is priced the same as the standard dLUTE SEQ reagent. For further information on the dLUTE SEQ DNA sequencing reagent please contact our support team or visit our dLUTE SEQ Forum.
dLUTE SEQ CF Ordering Details
We are currently developing a new formulation of dLUTE SEQ CF and until it is released we will no longer sell the older version.
The dLUTE SEQ CF DNA sequencing reagent is shipped directly from Australia by express courier. The estimated delivery times are:
- Australia – 2 working days
- Japan, Taiwan and South East Asia – 5 working days
- USA and Europe – 6 working days
- Rest of the world – 10 working days
Returns and Refunds Policy
Due to the delicate nature of our products, no refunds on any of our software or reagents can be be offered. Faulty goods, or goods damaged in transit, will replaced within 30 days of purchase. Please contact our support team for advice on product replacement issues.