2. Molecular

Molecular construction is the synthesize of your fragment, there's multiple ways to do this - including outsourcing if you can afford it



Obtain Insert:


First off, it may be possible that the plasmid you need is already created, check out the Plasmid Repositories in the Resources page.

Fragment synthesis is also available through companies such as Twist Bioscience if you want to skip the benchwork.

However, if purchasing isn't an option, then molecular construction is needed. If this sounds scary - worry not! I'm here to walk you through some options!



Note: In my opinion, the best option is Gibson/NEBuilder, skip ahead to this if you want to only learn one.


Build Construct:


Your main goal is to have your insert flanked with "sticky" ends - aka single stranded ends that are ready to anneal. These are combined with a linearized (aka cut) vector to make a complete plasmid/construct. 

There are so many more techniques than those which are in this presentation, but I go over the ones I think are important to know. If you're curious about another, look into recombinational/gateway which is another personal favorite of mine, I will likely update this blog to include it so stay tuned!





The four basic techniques that I'm going to cover are:

    • This is used to create short overhangs, your cassette must be flanked with two separate enzymes that are also in the plasmid 
      • using two separate enzymes allows for a unidirectional insertion instead of a combination of insertions
    • matching overlaps will be ligated to allow for the insertion and vector to become a complete plasmid


    • This will still need to be ligated together with ligase 

    • Simply make primers for your insert that will add an overlap to your backbone
      • The orange and green are the overlaps


    • There's two routes to use these overlapping primers
      • Mix
        • having two reactions- one for each overlap of the insert
          • Then simply combine, boil, and anneal; a portion of the result will have the insert with "sticky" ends
      • Incomplete
        • this method uses both overlap primers to make both at once, then simply cut the final extension step so that a small percentage of the product will have "sticky" ends after boiling and annealing


    • This will still need to be ligated together with ligase 

    • So this method uses T4 enzyme. This is an exonuclease as well as a polymerase, so you can use this polymerase to amplify your template with your overlap primers, but there's a catch - the overlapping portion cannot contain all 4 nucleotides, there has to be a limiting dNTP in your reaction.



  • Gibson Cloning/NEBuilder

    • Finally, time for Gibson!
    • No need, to make sticky ends before your reaction here, simply use your overlap primers to make your amplicon and get ready for the easy part


    • Remember this 3, 2, 1
      • 3 enzymes: T5 exonuclease (not nucleotide limited) will chew back the 3' ends of the linearized vector and the insert; ligase is used to combine your two+ fragments (insert and linearized vector); polymerase to fill in the gaps caused by the T5
      • 2+ fragments: insert and linearized vector. 
        • note: you can go above 2 as long as the overlaps are available.
        • A 3 part reaction is really common with Gibson: 2 fragments makeup the insert and 1 fragment is the backbone
      • 1 tube - that's right! All of this "magic" happens in a single tube reaction

🡲 E. coli

Last step here is the transition to the next stage. You have to get that complete plasmid of interest into bacteria to replicate rapidly. Luckily, certain strains of E. coli are really good at uptaking plasmids and replicating with them - we call these competent cells. You can make them...but I'd advise buying them :)

DH5 and 10 Beta are good starter cell lines, there are electro-competent and chemically competent cells. E.coli is typically used with chemical competency.

Follow the instructions in the kit you buy, but the general transformation process is as follows:



Ice your combination of plasmid and cells together for 20 minutes; heat shock for 30 seconds; ice again for 5 minutes. Add SOC outgrowth and either incubate or plate immediately depending on your bacterial resistance.







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