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SnapFast™ Cloning & Expression Vectors FAQs

Where do I find the sequences and maps of a SnapFast plasmid and insert?
How can I confirm the orientation of my insert if I use only one insertion site?
How have you removed unwanted restriction sites? Does it change the efficiency of translation?
What is the insert size capacity of the SnapFast system?
What should I do if I think the DNA I have bought is incorrect?
What are the fusion sites BsgI and BseRI?
Do you have any protocols to help with cloning?
Are your products IATA regulated and will you provide the required documentation?
How will my DNA be shipped and how much will be delivered?
What is your policy on Intellectual Property?
Is the SnapFast sytem patent protected?
How should I store my plasmid DNA?
If I don't see the vector I need, can Oxford Genetics make it for me?

 

Where do I find the sequences and maps of a SnapFast plasmid and insert?
The vector sequence can be found on the individual product pages for each plasmid in both the PDF and text files.

How can I confirm the orientation of my insert if I use only one insertion site?
We recommend, where possible, using the closest unique restriction sites that flank the DNA you want to insert. Because all our inserts are provided in the same backbone, pSF-Core, any sites that are external to the DNA insert you want to use should be compatible with your current vector, provided the insertion doesn't interfere with the gene or expression system you have already developed using our system.

If no unique sites are available then we recommend using the paired restriction sites that directly flank your insert. After ligation, restriction digestion of your clones should reveal a band size unique to your new inserted region. We are also developing primers that can be used to determine the orientation of any insert that is inserted into a single site by PCR, please contact us for further details on these primers if required. If you do not have these primers, we recommend sequencing a subset of those clones that are showing the presence of the correct band, this should determine your fragment's orientation. For a range of pre-designed sequencing primers please see the lower left menu on our site.

For many of our inserts, the orientation will not affect the insert function (e.g for bacterial selection markers and origins of replication), however, we recommend determining the orientation of your insert by sequencing to ensure sequence integrity and to allow you to build accurate plasmid maps for future research.

How have you removed unwanted restriction sites? Does it change the efficiency of translation?
The 20 naturally-occurring amino acids are encoded by 61 triplet codons, and most amino acids can be encoded by at least two different codons. Hence we can normally remove restriction sites from DNA sequences without changing the protein encoded. We routinely do this on the genes we sell and we always exchange for codons that are more frequently used in eukaryotes and prokaryotes where possible.

Most of the changes to the DNA sequences that we sell have been performed by either de novo DNA synthesis of a specific DNA sequence, or site directed mutagenesis. All of our genes are fully sequenced and tested for expression and activity before they enter our catalogue.

What is the insert size capacity of the SnapFast system?
In theory, there are very few restrictions. However, each vector does have a packaging limit that is dependent on the bacterial origin of replication you have inserted into your plasmid. If you wish to increase the size of your plasmid significantly you may wish to insert a lower copy number origin of replication or a bacterial artificial chromosome (BAC). To find the approximate upper limits of the origin of replication you currently have, please go to the product page for bacterial origins of replication.

The packaging or insert capacity of the SnapFast system is defined by the bacterial origin of replication in the particular plasmid you are using. For most research applications (up to around 10-15kb) a high copy pUC origin will work well. However, this origin of replication is unregulated by the bacterial cell and will produce hundreds on copies for each E.coli cell. This is beneficial in most plasmids because the DNA yield after plasmid extraction will be very high. However, this plasmid replication places a significant growth burden on the cell. As plasmid size increases this growth burden increases, and this can make plasmids unstable and produce low yields. By exchanging the plasmid origin of replication the growth burden can be reduced, allow larger plasmids to be created with better stability. In order of reducing copy number, our origins of replication are:

pUC Origin - 500-700 copies per cell

BR322 Origin - 15-20 copies per cell

p15A Origin - 10-12 copies per cell

SC101 Origin - 3-5 copies per cell

Note: The numbers are approximate but are a reasonable guide.

What should I do if I think the DNA I have bought is incorrect?
If you believe your DNA is incorrect then please contact us immediately. Where possible please try to include as much information about the tests you have performed, including any gel images and diagnostic restriction digests, and explain why you suspect there may be a mistake.

Please remember that restriction enzymes can expire and wear out. They can also produce STAR activity, leading to miscellaneous bands. Please try to confirm this is not the problem if possible.

What are the fusion sites BsgI and BseRI?
The fusion sites are a unique feature of the SnapFast system that allows you to fuse sequences to any gene that we sell in the main multiple cloning site. They work by cutting within the stop codon of the gene and allowing the direct ligation of a range of other DNA sequences to the gene as fusion coding sequences.

Do you have any protocols to help with cloning?
Yes, every plasmid page is linked to the Cloning Manual provided by Oxford Genetics.  In addition, there is a link to the “quick-start” protocol/guide to using your new vector.

Are your products IATA regulated and will you provide the required documentation?
All of our products are non-toxic, non-hazardous and non-infectious; hence they are not covered by any of the dangerous goods regulations. All plasmids are provided with a Safety Documentation Sheet (SDS) and the appropriate customs documentation.

How will my DNA be shipped and how much will be delivered?
Usually 5μg DNA is shipped in TE solution in a 2ml polypropylene tube, at ambient temperature.  You should receive your DNA in about 2 business days.

What is your policy on Intellectual Property?
Oxford Genetics operates an IP-friendly policy to maximise the usefulness for corporate customers. We claim no reach through rights on derivatives you may make using the plasmids we sell (unless you are a competing plasmid manufacturer, or unless we are obliged to because the plasmid contains someone else’s IP). See here for full details.

Is the SnapFast sytem patent protected?
Yes, but most of our vectors can be used for free by for-profit organisations.  We have patent pending on the SnapFast concept and system as well as the sequence of the core vector. Our patents are there to protect our materials from being re-created, or sold, by other organisations, not so that we can charge licence fees. Most of our vectors are free to use to produce reagents for profit (provided you are not a plasmid sales company). There are some exceptions where other third party organisations impose restrictions on the sequences we sell. When this is the case it will be clearly written on the detailed product sheet. If you have a specific question relating to intellectual property, please do not hesitate to contact Oxford Genetics directly.

How should I store my plasmid DNA?
DNA plasmids should be stored for long term stability at -20 °C and avoid multiple free thaw cycles. Keeping DNA at concentrations up to 1000 ng/µl at this temperature is common and freeze thawing should cause only minimal shearing of the DNA. However, samples that are higher in concentration (>1000 ng/µl) should ideally be diluted because ice crystal formation can damage the DNA at such high concentrations. Samples can be stored in water, elution buffer or Tris-EDTA (TE buffer). These buffers provide increasingly more stable conditions, but TE buffer can inhibit DNA sequencing reactions. 

When storing DNA at 4 °C, avoid using water as the storage buffer to reduce hydrolysis of the DNA. If storing for long term at 4 °C it is recommended that samples are simultaneously kept at -20 or -80 °C to ensure a stable sample is maintained. 

If a plasmid is kept at room temperature for 6 weeks, approximately a 2-3 fold drop in transformation efficiency will be observed. Room temperature storage is not recommended.

If I don't see the vector I need, can Oxford Genetics make it for me?
Yes, Oxford Genetics can design and build a vector to meet your specific needs. Find out more at Oxford Genetics - Sigma's partner for cloning and expression vectors for molecular biology and synthetic biology applications.