SnapFast DNA Assembly Master Mix is a high performance, low cost, single tube DNA assembly reagent that allows the efficient joining of up to 6 DNA molecules with homologous ends, without the use of restriction enzymes.
SnapFastTM DNA Assembly Master Mix is a high performance single tube DNA assembly enzyme mix that allows the efficient joining of any number of DNA molecules without restriction enzymes. DNA molecules with suitable homology (20 bp or more) can be joined efficiently in a single step reaction, and several molecules can be joined simultaneously to make long DNA molecules. SnapFast Master Mix is suitable for use with both long and short DNA sequences, and mixtures of long and short DNA molecules can be joined simultaneously.
SnapFastTM Master Mix Benefits:
Cost Per Reaction
SnapFastTM DNA Assembly Master Mix has been designed for both small scale and high-throughput applications, with a low cost per reaction when compared to other cloning systems.
* To work out equimolar amounts DNA, work out the lengths of your fragments in base pairs. Then work out how much longer each one is compared to the other. For example, if you are joining a 500 bp fragment to a 5000 bp linearised plasmid, you know that the fragment is 10 times smaller than the plasmid. Therefore, to get the same number of DNA molecules in your reaction (equimolar) you simply have to use 10 times more plasmid in nanograms compared to your fragment because the DNA mass on a per molecule level is proportional to its length. So in this example you might use 10 ng of fragment compared to 100 ng plasmid (1:10 ratio). Dy doing this half of the DNA molecules will be the fragment and the other half will be the plasmid.
** When joining more that 2-3 DNA fragments together higher efficiency can be gained by using a higher quantity of DNA (>100 ng). Higher efficiency may also be gained by using additional SnapFastTM Master Mix.
This reagent has no IP restrictions and can be used for both non-commercial and commercial cloning applications.
This kit contains a single tube enzyme mix (110 microlitres per 1.5ml polypropylene tube) that allows DNA fragments bearing homologous ends to be joined. The specific components of the reagent are proprietary.
This reagent should be stored at -20 degree Celsius. The reagent freezes at this temperature. To prevent loss of enzyme activity, avoid multiple freeze thaw cycles.
This reagents activity is confirmed by transforming two fragments (one containing a bacterial expression plasmid and another containing GFP) and confirming DNA assembly by counting GFP positive colonies. We also repeat this process for the same construct but where it has been split into 5 fragments.
Why use SnapfastTM Master Mix over traditional enzyme based cloning techniques?
Using SnapfastTM Master Mix over traditional enzyme based cloning techniques gives the user more control over the final sequence allowing for less unwanted additional sequence after the DNA has been assembled. Due to the use of fragment(s) with Homologous ends to each other and the vector they can be inserted into any positions of a linearized vector. Compared with enzyme based cloning it is a much faster method with a higher efficiency for assembling multiple fragments.
What are the upper and lower size limits for assembling DNA fragments?
This reagent can be used to clone large fragments, above 15Kb into any plasmid. For long fragments of 5Kb or longer we recommend using longer regions of homology, ideally over >30bp.
How many fragments can be assembled at the same time?
Up to 6 fragments can be assembled in one reaction, the more fragments that are joined together simultaneously the lower the efficiency will be.
What are the smallest homologous regions that can be used for assembly?
The recommended homologous regions of at least 15bp, smaller regions can be used but this may reduce the efficiency of DNA assembly.
What are the longest homologous regions that can be used for assembly?
The reagent allows for the assembly of fragments with homologous regions of up to 150bp in length.
What are the recommended storage conditions for SnapfastTM Master Mix?
It is recommended that SnapfastTM Master Mix is stored at -20°C, multiple thaw freeze cycles should be avoided where possible as it may denature the enzymatic content.
Is inactivation of restriction enzymes required after vector digestion?
Inactivation is only required if the fragment(s) contain any of the same restriction sites as the enzymes used. If the restriction enzymes used are heat resistant then purification should be carried out using DNA columns, phenol-chloroform extraction or extracted from agarose gel after electrophoresis before being used in a SnapfastTM Master Mix reaction.
Will other temperatures work for SnapfastTM Master Mix reactions?
SnapfastTM Master Mix has been optimised for use at 50°C.
Can I use a small repeating sequence as my homologous regions such as a repeating tag?
Small repeating sequences like tags should be avoided in the homologous regions as it may reduce efficiency and hinder recombination. To avoid this, add at least 3 nucleotides at the end of a repeating sequence.
Will the product of a SnapfastTM Master Mix reaction withstand PCR amplification?
Due to final product being covalently bonded it will withstand PCR amplification.
How competent do the cells need to be for transforming a SnapfastTM Master Mix reaction?
It is recommended that competent cells that produce more than 106 CFU/µg DNA are used for chemical transformation. Using cells that are competent enough is very important, for any cloning that yields low efficiency it is suggested to use more efficient competent cells or a different type.
Can an electroporation protocol be used instead of chemical transformation?
Yes, follow the protocol guidelines that come with the electrocompetent cells used.
What can I use for a control reaction?
As a control reaction, cut or PCR amplified plasmid can be used with SnapFastTM Master Mix. This provides the background level of uncut plasmid and illegitimate re-circularising during the reaction (the latter should not happen with this reagent, but this sequence dependent and assumes the ends of your vector have no homology).