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Classical cloning with FastDigest Restriction Enzymes

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  • Classical cloning with FastDigest Restriction Enzymes

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    There are several types of cloning technologies available to produce recombinant clones for the molecular cloning workflow. Thermo Scientific™ FastDigest™ restriction enzymes are a traditional cloning technology for obtaining high-quality cloned DNA.

    Found naturally in bacteria, restriction enzymes recognize and cleave specific DNA sequences, resulting in sticky ends (5ʹ or 3ʹ protruding ends) or blunt ends, enabling DNA inserts to be cloned into vectors with compatible ends. Star activity, buffer compatibility, and incomplete digestion are some common hurdles in restriction digestion.

    Simplify your restriction enzyme workflow

    FastDigest™ enzymes are an advanced line of restriction enzymes that offer fast and complete digestion of DNA in a single, universal buffer. FastDigest™ enzymes save time and effort while increasing throughput.

    · Complete digestion in 5-15 minutes
    · 176 unique specificities
    · 100% buffer compatibility with downstream applications
    · No star activity due to short incubation times
    · Direct loading of reaction mixture on gels

    100% activity in a single buffer

    The FastDigest™ Green Buffer and Thermo Scientific™ FastDigest™ Buffer are proprietary digestion buffers which support 100% activity of all FastDigest™ restriction enzymes. This system allows for double and multiple digestions with any combination of enzymes. No sequential digestions or buffer changes are needed.

    100% buffer compatibility with downstream applications

    DNA/RNA modifying enzymes, such as ligases, phosphatases, kinases and mesophilic DNA polymerases have 100% activity in FastDigest™ and FastDigest™ Green Buffer. Therefore, enzymes used in downstream applications can be directly added to the FastDigest™ reaction mix. No buffer changes or purification steps are needed.

    Explore our wide range of FastDigest™ Restriction Enzymes at

    Type IIS Restriction Enzymes

    Type IIS restriction enzymes comprise a specific group of enzymes which recognize asymmetric DNA sequences and cleave at a defined distance outside of their recognition sequence, usually within 1 to 20 nucleotides. This specific mode of action of Type IIS restriction enzymes is widely used for innovative DNA manipulation techniques, such as Golden Gate cloning, enabling sequence-independent cloning of genes without the need to modify them by including compatible restriction sites (scars).

    Benefits of cloning with Type IIS restriction enzymes

    · Single-tube cloning—digestion and ligation reactions can take place in the same tube at the same time because the restriction site is eliminated from the ligated product.
    · Scarless cloning—scar sequences are not being introduced because the overhang sequence created is not dictated by the restriction enzyme
    · Assembly of multiple fragments—multiple inserts can be assembled together simultaneously by using the right combination of complementary ends.

    Thermo Fisher Scientific™ offers ten Type IIS restriction enzymes within the Thermo Scientific™ FastDigest™ restriction enzyme portfolio, discover more at

    FastDigest™ Online Selection Tool

    To find the right FastDigest™ enzyme for your downstream application browse our online selection tool. You can easily search by product name, isoschizomer name, recognition sequence, or catalog number.

    FastDigest™ Enzyme Selection Tool



    Get a FastDigest™ Value Pack

    Try the top 13 enzymes, and both the colorless and green FastDigest™ Buffers, get our Thermo Scientific™ FastDigest™ Value Pack (Cat. No. K1991).



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  • Directional Cloning | Directional Cloning Vs Traditional Cloning |

    2:07

    DNA insert and vector molecules are digested with two different restriction enzymes to create noncomplementary sticky ends at either end of each restriction fragment. This allows the insert to be ligated to the vector in a specific orientation and prevent the vector from self-ligation.   

    In easy way, directional cloning generally refers to the ligation of an insert into a vector in a known orientation.

    The most commonly used method is to cut the 5' and 3' sites on the vector and insert with different restriction enzymes that yield different sticky ends. This ensures that when you ligate insert into vector, the different identity of the 5' and 3' sticky ends ensures only 1 possible orientation for the insert to effectively anneal to the vector.

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  • Thermo Scientific Double Digestion Day

    2:11

    Double digest your DNA in only 5-15 minutes with one buffer and load directly onto your gel with 176 Thermo Scientific FastDigest Enzymes.

    Thermo Scientific FastDigest enzymes are an advanced line of restriction enzymes that create a new standard in DNA digestion. During our 30 years of restriction enzyme research, we compiled one of the largest collections of restriction enzyme producing bacterial strains in the industry. Our large section of enzyme isoschizomers and state-of-the-art production facility facilitated the creation of the unique system of 176 FastDigest® restriction enzymes.

    Find out more at

  • Restriction Enzymes 2

    4:07

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  • Restriction Enzyme Reaction

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  • Cut Up Parody Music Video

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    Full lyrics:
    Cut, cut, cut, cut
    Restriction enzymes cut sequences in DNA
    BspHI cuts the seq TCATGA
    If it leaves sticky ends, that’s called single stranded overhang
    Single stranded tails bind other complementary chains
    2 strands will ligate if they’re cut by the same (enzyme)
    Non-complementary strands won’t bind -- I know that's lame!
    Blunt ends, got no tail, it could bind to anything
    SmaI cuts straight down, no single stranded overhang
    Sticky ends got single chains, blunt ends got no thang
    Let the enzymes do they thang, restriction sites in everything

    If it cut, if it cut, if it cut, sticky or blunt
    If it cut, if it cut, if it cut, sticky or blunt
    If it cut, then it cut. If it cut, then it cut.
    If it cut, if it cut, if it cut, sticky or blunt

    Fragments different sizes, how to tell what size this is?
    Gel electrophoresis shows the size, no surprises.
    Pour the gel, then don’t touch, let the liquid dry, sis
    No more comb or rubber stoppers, now you know what time it is
    Pour the buffer on the gel, DNA? Can’t see it still
    Add a little bit of loading dye so we can see it well
    Use a steady hand to put the samples iin
    Plug it in to power cell, black to black and red to red

    Watch the samples move real slow, leave it 60 mins or so
    Voltage stays on the low, don’t wanna bump it tho
    DNA is charged -- Negative to positive flow
    Peek at the side for bubbles forming-- Now thas cool, yo!
    Take the gel out, place it on a UV light
    Each band on the gel is DNA glowing bright
    Take a picture, print it out, on the paper, we gon write
    Each band’s a certain size, cut at restriction sites

    If it cut, if it cut, if it cut, sticky or blunt
    If it cut, if it cut, if it cut, sticky or blunt
    If it cut, then it cut. If it cut, then it cut.
    If it cut, if it cut, if it cut, sticky or blunt

    Bands at the top are big, bands that are below are small,
    Big pieces move slow, easier for bits to fall.
    One sample ran should have sizes that are known
    Compare the distance travelled of the other bands that’s shown.
    Bands at the same height are likely the same size.
    Estimate the size, probably… 995.
    If a band falls in between, then read between the lines
    Not the most accurate, just estimation by design

    Take the gel out, place it on a UV light
    Each band on the gel is DNA glowing bright
    Take a picture, print it out, on the paper, we gon write
    Each band’s a certain size, cut at restriction sites

    If it cut, if it cut, if it cut, sticky or blunt
    If it cut, if it cut, if it cut, sticky or blunt
    If it cut, then it cut. If it cut, then it cut.
    If it cut, if it cut, if it cut, sticky or blunt

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    Suppose We have stertch of DNA containing the traget gene to be clonned.

    The central feature of restriction enzyme is that it has a particular recognition sequence at which it cuts a DNA moleule.

    Here we are taking example of EcoR1 ...

    So what this enzyme will do is that basically it would scan all over the dna until it finds this specific sequence.
    And wherever this particular sequence is found in the dna it would cleave the dna at that point.

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