Gateway Cloning: Simple and Multisite Gateway Cloning - LR and BP Cloning

00:14:41
https://www.youtube.com/watch?v=LpjqgT-WKgY

Résumé

TLDRDenne video gennemgår Gateway kloning, som er en metode til kloning af DNA uden restriction enzymer, ved hjælp af site-specifik rekombination. Videoen forklarer forskellen mellem Gateway kloning og traditionelle kloningsmetoder, uddyber BP og LR reaktioner i Gateway kloning, og præsenterer både simple og komplekse kloningsmetoder. Der diskuteres også fordele og ulemper ved Gateway kloning, herunder tidseffektivitet og potentielle problemer med ekspressionsvektorer.

A retenir

  • 🔬 Gateway kloning er en effektiv metode til DNA kloning uden restriction enzymer.
  • 🔄 BP reaktioner muliggør rekombination mellem donor DNA og vektorer.
  • 🔧 LR reaktioner involverer integration og excision af DNA.
  • ⚗️ Multi-site Gateway kloning kan håndtere flere DNA-fragmenter på én gang.
  • 🧬 Ingen restriktion enzymer betyder scarless kloning, men kræver stadig donor vektorer.
  • 💰 Høje omkostninger ved at skifte fra Gateway kloning til andre metoder.
  • ⚠️ Uønskede sites kan påvirke ekspression i Gateway vektorer.
  • 🗂️ Univector metoder er et alternativ til Gateway ved brug af locks og spe sites.

Chronologie

  • 00:00:00 - 00:05:00

    Videoen introducerer Gateway-kloning og dens forskelle fra klassisk restriktionsenzym-baseret kloning. Gateway-kloning kræver ikke restriktionsenzymer og erstatter de laborintensive trin i klassisk kloning med hurtigere rekombinationstrin. Ved at bruge specifikke attachment sites (at sites) i en BP-reaktion, kan DNA fra donorvektorer hurtigt integreres i målvektoren, hvilket muliggør en mere effektiv kloningsproces.

  • 00:05:00 - 00:14:41

    Denne del af videoen går i dybden med de skridt, der er involveret i Gateway-kloning, inklusive de komplekse aspekter af multipla Gateway-strategier, hvor flere DNA-segmenter kan klones samtidigt. Selvom Gateway-effektivitet mindsker tiden for kloning, indebærer det nogle udfordringer, som f.eks. at skabe donorvektorer med specifikke at sites og potentielle problemer med udtryk af gener på grund af indbyggede restriktionssteder. Generelt fremhæver videoen, at Gateway-kloning er en kraftfuld metode, men også en der kræver omhyggelig planlægning og forståelse af de involverede processer.

Carte mentale

Vidéo Q&R

  • What is Gateway cloning?

    Gateway cloning is a method for cloning DNA without using restriction enzymes, utilizing site-specific recombination instead.

  • How does Gateway cloning differ from traditional cloning methods?

    Traditional cloning involves restriction enzymes to cut DNA and ligate fragments, while Gateway cloning simplifies the process by using recombination.

  • What are BP and LR reactions in Gateway cloning?

    BP reactions involve the exchange between attachment sites on the vector and donor DNA, while LR reactions facilitate excision and integration of the viral DNA.

  • What are the advantages of Gateway cloning?

    Gateway cloning saves time, increases efficiency, allows for high throughput, and enables scarless cloning.

  • What are some disadvantages of Gateway cloning?

    It requires initial cloning into donor vectors and can have undesirable sites within the expression constructs.

  • What is multi-site Gateway cloning?

    Multi-site Gateway cloning allows simultaneous cloning of multiple DNA fragments into a single vector.

  • What are the attachment sites involved in Gateway cloning?

    The attachment sites include at P and at B for the Gateway system.

  • Can Gateway cloning be used with restriction enzymes?

    Yes, initial cloning steps may still require restriction enzymes to create the donor vectors.

  • What is a univector cloning method?

    Univector cloning is another approach that uses locks and spe sites for cloning, similar to Gateway.

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Sous-titres
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  • 00:00:00
    in this video we will see what is
  • 00:00:02
    Gateway cloning and how it is different
  • 00:00:04
    from classic restriction enzyme based
  • 00:00:07
    cloning I will go over some basic
  • 00:00:09
    knowledge and Link it to simple Gateway
  • 00:00:11
    cloning strategy as well as complex
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    Gateway methods I will discuss specifics
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    of LR and BP reactions and dive into
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    their steps in a bit of detail finally
  • 00:00:24
    we will see what makes Gateway great and
  • 00:00:26
    some reasons to stay away from it
  • 00:00:28
    Gateway cloning has been around for more
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    than two decades and is commercialized
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    by invitrogen this means you can buy it
  • 00:00:34
    as a kit it is a method to clone without
  • 00:00:37
    using restriction enzymes traditionally
  • 00:00:40
    in restriction based cloning you start
  • 00:00:42
    with a vector a plasmid backbone into
  • 00:00:45
    which you would like to insert something
  • 00:00:47
    so you digest and perhaps purify the
  • 00:00:49
    vector in parallel you prepare the
  • 00:00:51
    insert which could be a PCR product or
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    perhaps a piece of DNA from another
  • 00:00:56
    Vector which means you are subcloning
  • 00:00:59
    depending on your strategy you digest
  • 00:01:01
    maybe you blunt and purify the insert
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    and combine it with the backbone this
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    liation gets transformed selected
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    screened and eventually validated
  • 00:01:10
    possibly for orientation if you do blunt
  • 00:01:12
    cloning I have separate video that
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    discusses these things in much more
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    detail these steps from start to the
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    liation are labor intensive Gateway
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    cloning replaces all these upstream
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    manipulations and liation steps in one
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    super quick reaction so what is
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    happening in this Gateway cloning relies
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    on at site recombination at sites are
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    attachment sites and they are DNA
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    sequences here's some context to
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    attachment sites think of a happy
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    bacteria that is swimming but out of
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    nowhere a bacteria phage Lambda attacks
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    the bacteria the infection leads to
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    viral DNA injection into the bacteria if
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    you know a little bit about the virus
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    life cycle the viral DNA can integrate
  • 00:01:57
    into the bacterial genome this
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    integration occurs due to recombination
  • 00:02:01
    of the attachment sites between the two
  • 00:02:03
    genomes so both virus and bacteria DNA
  • 00:02:07
    carry at sites Fage at sites are called
  • 00:02:10
    at P bacterial at sites are called at B
  • 00:02:14
    depending on how you define them they
  • 00:02:16
    range from 25 to 300 nucleotides in
  • 00:02:19
    length a typical atsite is split into
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    two portion say p and p Prime and the
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    middle recombination spot is called o
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    likewise you have B and B Prime and the
  • 00:02:31
    middle spot is called o the O is the
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    recombination spot between at p and at B
  • 00:02:37
    let's actually understand how this viral
  • 00:02:39
    integration works and how at site
  • 00:02:41
    recombination is involved in this the
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    virus DNA as we said has the ATP site
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    I'm drawing the virus DNA as circular
  • 00:02:49
    for ease of understanding instead of
  • 00:02:51
    calling these two halves of ATP as p and
  • 00:02:54
    p Prime let's name them one and two the
  • 00:02:57
    ATP of pectoral DNA likewise has has B
  • 00:03:00
    and B Prime but let's call them three
  • 00:03:02
    and four when the viral DNA needs to
  • 00:03:04
    integrate into the host genome it makes
  • 00:03:07
    an enzyme called integrace the Integra
  • 00:03:10
    is recognize at B and at p and cause a
  • 00:03:13
    recombination to occur between them this
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    recombination between the two ad sites
  • 00:03:18
    is called a BP reaction recombination
  • 00:03:21
    event occurs at the O region between the
  • 00:03:23
    two at sites if you start from the
  • 00:03:26
    perspective of site three the
  • 00:03:28
    recombination brings number number two
  • 00:03:30
    in front of three and now this entire
  • 00:03:33
    viral DNA until number one comes along
  • 00:03:35
    with number two but now the number one
  • 00:03:38
    gets attached to number four from number
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    four we get back to the bacterial DNA
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    which comes full circle to number three
  • 00:03:46
    from this BP reaction we see that the
  • 00:03:48
    virus DNA has integrated into the
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    bacterial DNA and that is the proage
  • 00:03:53
    life cycle of the virus also just to get
  • 00:03:56
    back to these chimeric BP sites this BP
  • 00:03:59
    site is called at L and this PB site is
  • 00:04:03
    called at R these stand for left and
  • 00:04:07
    right you may come across some old
  • 00:04:09
    literature that calls them Bop or po but
  • 00:04:13
    I will use at L and at R terminology now
  • 00:04:17
    that we understand this let's look at a
  • 00:04:19
    simple Gateway cloning this uses 2 ATP
  • 00:04:22
    and 2 at B sites assume that our final
  • 00:04:25
    Vector will be this blue Vector carrying
  • 00:04:28
    a conomy marker it carries two at P
  • 00:04:31
    sites with a ccdb suicide gene in the
  • 00:04:33
    middle the second Vector carries two at
  • 00:04:36
    B sites and it has the DNA of Interest
  • 00:04:39
    we want to clone in the middle this is a
  • 00:04:41
    donor Vector just like this DNA of
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    Interest being in the plasmid form you
  • 00:04:46
    can also have the DNA of interest from a
  • 00:04:48
    PCR but in that case you will have to
  • 00:04:50
    add add B sites to the primer for
  • 00:04:53
    Simplicity I will restrict the
  • 00:04:54
    discussion to the plasmid form of the
  • 00:04:56
    donor Vector for Gateway cloning you
  • 00:04:58
    take these two plas and combine them
  • 00:05:00
    with integrases in a BP reaction this
  • 00:05:04
    causes recombination between the ATP and
  • 00:05:06
    at B sites so now we can redraw the
  • 00:05:09
    final vector and let's start with this
  • 00:05:11
    part of at P which gets switched with at
  • 00:05:14
    B and then the DNA I continues until you
  • 00:05:16
    have half of at B which gets recombined
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    with ATP and this comes back full circle
  • 00:05:24
    essentially we have swapped DNA into the
  • 00:05:27
    final Vector like this you can also we
  • 00:05:29
    draw the donor Vector which instead of
  • 00:05:31
    DNA I now contains the ccdb Gene and
  • 00:05:35
    like we saw earlier you can annotate
  • 00:05:37
    these new chimeric sites as at R and at
  • 00:05:41
    L note that all of this happens in vitro
  • 00:05:44
    and following this reaction it is ready
  • 00:05:46
    to be transformed and the bacteria is
  • 00:05:48
    grown in kyin because the final Vector
  • 00:05:51
    containing our DNA I has kyin which
  • 00:05:54
    means any bacteria carrying this other
  • 00:05:57
    plasmid will die so in in this simple
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    Gateway cloning we have learned about
  • 00:06:02
    one simple rule at p and at B can
  • 00:06:05
    recombine but at p and at P do not
  • 00:06:09
    recombine likewise at B and another at B
  • 00:06:12
    cannot recombine if these two were true
  • 00:06:15
    then bacterial and viral DNA during
  • 00:06:17
    infection process will recombine by
  • 00:06:19
    themselves and that will mess up
  • 00:06:21
    everything all right so we have switched
  • 00:06:24
    this internal portion between the two
  • 00:06:25
    plasmids and this is a recombination
  • 00:06:28
    mediated exchange but we saw that during
  • 00:06:32
    infection the viral DNA integrates into
  • 00:06:34
    the host genome but in Gateway cloning
  • 00:06:37
    we talk about exchange so where is the
  • 00:06:40
    integration event in this Gateway
  • 00:06:43
    process let's break this process down
  • 00:06:45
    and understand stepwise how integration
  • 00:06:48
    can lead to exchange we have two at p
  • 00:06:52
    and two at BS for Simplicity let's label
  • 00:06:55
    the halves as 1 2 3 4 5 and 6 and 7even
  • 00:06:58
    and 8 and to to make this simple imagine
  • 00:07:01
    that this pair of at gets recombined
  • 00:07:03
    first Also let's assume that this second
  • 00:07:06
    pair is not used until the first
  • 00:07:08
    reaction is complete and we will focus
  • 00:07:10
    on the final vector and anchor at this
  • 00:07:13
    site one as we saw before anchor site 1
  • 00:07:16
    recombines with site six site 6
  • 00:07:19
    continues into site 7 and site 8 if we
  • 00:07:22
    continue we see this entire plasmid
  • 00:07:25
    comes back to site five site five gets
  • 00:07:28
    recombined with site two and this site
  • 00:07:30
    two continues to site three and four and
  • 00:07:33
    all this circle backs to chomy this is
  • 00:07:36
    an intermediate plasmid where only one
  • 00:07:39
    BP reaction has occurred and we have
  • 00:07:41
    integrated the donor Vector into the
  • 00:07:43
    final Vector in this we have one at r
  • 00:07:47
    one at L one at B and one at P the at B
  • 00:07:52
    and P can further undergo a second BP
  • 00:07:55
    reaction to understand this next step it
  • 00:07:58
    is best to redraw the plasmid in a
  • 00:08:01
    different configuration this is done
  • 00:08:03
    such that the B and P are next to each
  • 00:08:05
    other you can pause this video and try
  • 00:08:08
    to draw this yourself as well in the
  • 00:08:10
    second BP reaction the site 7 gets
  • 00:08:13
    recombined with site 4 which comes back
  • 00:08:15
    full circle to conomy this releases our
  • 00:08:19
    dnf interest in the conomy plasmid the
  • 00:08:22
    second recombination occurs from site
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    three to site 8 which Circle backs to
  • 00:08:27
    illin with the chimeric site 5 and 2 and
  • 00:08:30
    the ccdb gene you can label all these
  • 00:08:33
    sites as at R and at L if you look
  • 00:08:37
    closely these are exactly the same
  • 00:08:39
    product we had originally produced so
  • 00:08:41
    this way sequential BP reactions can
  • 00:08:44
    integrate and exchange the DNA with ccdb
  • 00:08:47
    in the donor and the final Vector okay
  • 00:08:50
    now we can move to the second part of
  • 00:08:52
    the simple Gateway cloning you remember
  • 00:08:55
    this proage structure we discussed a few
  • 00:08:57
    minutes ago where you get the full at L
  • 00:09:00
    and at R another aspect of the virus
  • 00:09:03
    life cycle is that propage can move to a
  • 00:09:05
    litic cycle where it is possible that
  • 00:09:07
    the viral genome is excised out of the
  • 00:09:10
    bacterial genome how does that happen to
  • 00:09:13
    understand that we have to redraw the
  • 00:09:15
    configuration of this genome in the
  • 00:09:17
    following way during litic phase some
  • 00:09:19
    exision integrases and other enzymes are
  • 00:09:22
    expressed these enzymes can recombine at
  • 00:09:25
    R and at L looking at site three it will
  • 00:09:28
    recombine with four and this gives you
  • 00:09:31
    the bacterial DNA likewise number one
  • 00:09:33
    recombines with number two which
  • 00:09:35
    releases the viral DNA previously we
  • 00:09:38
    have noted BP reaction that occurs
  • 00:09:40
    between at B and at P this excision
  • 00:09:43
    occurs between at L and at R so we call
  • 00:09:47
    this an LR reaction from the LR reaction
  • 00:09:51
    we can recover the at p and at B sites
  • 00:09:54
    just like B and P we can have another
  • 00:09:57
    variant in our simple Gateway cloning
  • 00:09:59
    where we use two at R and two at l s
  • 00:10:02
    sites in this style of Gateway the final
  • 00:10:05
    Vector has at l s sites for example and
  • 00:10:08
    likewise the donor Vector will have
  • 00:10:10
    compatible at R sites and these two
  • 00:10:13
    vectors will use another set of specific
  • 00:10:15
    enzymes and this will be an LR reaction
  • 00:10:19
    and the recombination occurs in a
  • 00:10:20
    similar fashion except that the final
  • 00:10:22
    forms have ADD B and add p in the
  • 00:10:25
    products this is yet another example of
  • 00:10:28
    recom combination mediated exchange the
  • 00:10:32
    rules for LR reactions are that L and R
  • 00:10:35
    can recombine but two L's cannot and
  • 00:10:38
    likewise two at RS cannot the
  • 00:10:41
    interesting bit to note is that the at B
  • 00:10:43
    containing DNA I in the final Vector can
  • 00:10:46
    become a new donor for a new BP reaction
  • 00:10:50
    if you wanted to use it now that you
  • 00:10:52
    understand the simple Gateway cloning
  • 00:10:54
    let me give you a snapshot of some
  • 00:10:56
    complex ways to do Gateway cloning this
  • 00:10:58
    is called a multi-site Gateway simple
  • 00:11:02
    Gateway has at B and at P compatibility
  • 00:11:05
    however there are variants of at B and
  • 00:11:07
    PS for instance you have at B1 and at P1
  • 00:11:11
    which are compatible with each other but
  • 00:11:13
    B and P1 are not compatible and likewise
  • 00:11:16
    B1 and P are not compatible just like B1
  • 00:11:20
    and P1 you also have other variants on B
  • 00:11:22
    and PS suffice to say that their
  • 00:11:24
    products are likewise also variants and
  • 00:11:27
    one variant pair is not compatible with
  • 00:11:30
    another variant pair let's do this with
  • 00:11:33
    an example where you're trying to clone
  • 00:11:35
    a promoter and an ORF into a plasmid at
  • 00:11:38
    the same time let's assume that the
  • 00:11:40
    final Vector has at R1 and at R2 then
  • 00:11:43
    you have the two donors one containing
  • 00:11:46
    the promoter between at L1 and at R3 and
  • 00:11:50
    the second donor containing the ORF
  • 00:11:52
    between L2 and L3 in the multi-site
  • 00:11:56
    Gateway you mix all these three vectors
  • 00:11:58
    in one reaction and cross Vector
  • 00:12:00
    recombination can occur in the presence
  • 00:12:03
    of specific LR reaction enzymes each
  • 00:12:06
    variant gets recombined independently if
  • 00:12:09
    you solve this puzzle step by step you
  • 00:12:11
    will note that the promoter flanked by
  • 00:12:13
    B1 and B3 the ORF is flanked by B3 and
  • 00:12:17
    B2 and this entire configuration is
  • 00:12:21
    present in the final canamy inor if you
  • 00:12:24
    want step-by-step details on this
  • 00:12:26
    reaction it is available on my patreon
  • 00:12:28
    along with the reference material used
  • 00:12:30
    to make this video this multi-site
  • 00:12:32
    Gateway doesn't just stop here you can
  • 00:12:35
    come in with a new Vector containing P1
  • 00:12:37
    P2 and P3 and switch out the ORF if you
  • 00:12:40
    want it to or even the promoter using a
  • 00:12:43
    donor Vector containing P1 and P3 this
  • 00:12:46
    brings us to the last segment of this
  • 00:12:48
    video the advantage of Gateway is that
  • 00:12:51
    it saves time but this comes at a very
  • 00:12:53
    high cost but it is quite efficient it
  • 00:12:56
    reaches very high throughput and it is
  • 00:12:58
    very flexible as we saw from the
  • 00:13:00
    multi-site Gateway the process is
  • 00:13:03
    virtually scarless since no restriction
  • 00:13:05
    enzymes are involved multi-site Gateway
  • 00:13:08
    is also very powerful because it saves
  • 00:13:10
    you sequential cloning steps if you were
  • 00:13:13
    to use classic enzyme based cloning
  • 00:13:15
    methods the disadvantage is that
  • 00:13:18
    restriction enzymes are not involved so
  • 00:13:20
    you're actually stuck in this Gateway
  • 00:13:22
    cloning ecosystem and the cost of
  • 00:13:24
    switching out is very high we did say
  • 00:13:27
    that the lack of restriction enzymes
  • 00:13:29
    leaves no scar but you still have to
  • 00:13:31
    clone your DNA into a donor Vector with
  • 00:13:34
    at sites so it is not 100% enzyme free
  • 00:13:38
    this becomes very complicated if you
  • 00:13:39
    were starting from a scratch and you
  • 00:13:41
    have to build multiple donor vectors
  • 00:13:43
    containing these complex sites so
  • 00:13:46
    restriction enzymes are still required
  • 00:13:48
    in the beginning stages of the Gateway
  • 00:13:50
    cloning there's one concern with Gateway
  • 00:13:52
    expression vectors like the one here
  • 00:13:54
    containing a promoter and an ORF there
  • 00:13:57
    is this undesirable ad B site you find
  • 00:14:00
    in the middle of the ORF and promoter
  • 00:14:02
    and it may be that it interferes with
  • 00:14:05
    the expression Dynamics these are some
  • 00:14:07
    points I would like you to consider when
  • 00:14:09
    it comes to Gateway cloning so ATP and B
  • 00:14:12
    are involved in Gateway a similar
  • 00:14:15
    ecosystem of cloning is called uni
  • 00:14:17
    vector which works like Gateway but it
  • 00:14:20
    relies on locks spe sites so both of
  • 00:14:22
    these are recombination mediated
  • 00:14:24
    cassette exchange cloning methods there
  • 00:14:27
    is an orthogonal approach roach that
  • 00:14:29
    uses homologous recombination some
  • 00:14:32
    methods in this space are infusion magic
  • 00:14:35
    sefc perhaps you will see them in a
  • 00:14:38
    separate video and maybe I will see you
  • 00:14:40
    there as well
Tags
  • Gateway cloning
  • retriction enzymes
  • recombination
  • DNA
  • cloning methods
  • BP reactions
  • LR reactions
  • multi-site Gateway
  • attachment sites
  • univector