General Cloning Protocol

=Cloning= ''This page seeks to provide an introduction to cloning techniques and methodologies for the beginning researcher and assumes ready knowledge in molecular biology. iGEM students, starting UROPS, and people who have worked in labs but simply have never had to deal with cloning before may find it a useful addition to what they find in their textbooks.''

Introduction
"Cloning" is the term used in molecular biology for the insertion of another organism's gene into a target host organism (eg. taking the Green Fluorescent Protein (gfp) gene from the A. victoria jellyfish and putting it in E. coli to get E. coli to glow green). Though this sounds simple, multiple in vitro DNA-assembly steps are involved that are not always simple in execution. One should remember that each of these in vitro steps is in essence a chemical reaction (often carried out by enzymes), which is subject to environmental conditions (salt concentration, temperature), quality and quantity of reagents (how much cut DNA, presence of dNTP's, etc), and theoretical yields. If the yield for any of these reactions in our series is too low, then we are unlikely to get the desired end product (usually our gene of interest inserted into our target plasmid). Therefore, we should always seek to optimize the yield of desired product for each of our reactions; this simply means use the HIGHEST quality reagents in the most OPTIMAL conditions you can. Here is a highly generalized set of steps in cloning: Depending on what one wants to build, the construction steps for cloning can look very different. Even for identical constructs, differences in style among researchers will lead to different cloning methodologies. If one chooses to stick to standardized systems of cloning such as BioBrick cloning, construction protocols can be identical across almost all situations. Such standardized systems can greatly simplify how one approaches cloning, but they are not always suited to every situation (eg. standard assembly in BioBrick cloning runs into problems when one wants to build fusion peptides). The take-home message here is that there is rarely ONE WAY in which to clone something. How we build DNA constructs depends on what we need to build and how the individual researcher wants to build it. Things people take into account in planning the assembly are: cost, speed, reliability, accuracy. Arguably, synthesis technology takes the cake on speed, reliability, and accuracy, but at even 70cents/bp, it is still prohibitively expensive for most people's cloning needs.
 * 1) Production of the linear insert. A linear piece of a desired DNA sequence can be obtained in many ways, including traditional PCR, Assembly PCR, cutting a piece out of an already existing vector (as in biobricks cloning), synthesis orders (through companies), primer extension reactions, and  primer annealing.
 * 2) Cutting the insert and target vector with appropriate endonucleases. We use restriction endonuclease enzymes to cut specific sequences of DNA. The ends of these cut pieces of DNA will then stick together if their overhang sequences are complementary, allowing us to ligate linear pieces of DNA together in a highly specific way.
 * 3) Ligating the linearized vector and insert together. Here, we combine our insert and vector in a reaction with the enzyme DNA ligase, which covalently links free ends of DNA together.
 * 4) Transformation of the completed vector and screening. E. coli will take up DNA through its membranes under certain conditions (heat shock, electroporation), which allows us to put our finished DNA vector into the bacterium. We plate our transformants on selective media, which will allow only those cells to grow that have successfully taken up our vector.

A more detailed example:
What follows is a highly specific example of a cloning protocol. Let's say you want to assemble two genes, pepA and gfp, in series. When the construction is complete, you would like the products of genes pepA(300bp) and gfp(800bp) to be the fused protein PEPA-GFP, with a 2xGGGS linker in between the two domains. You also want to add: a medium-strength promoter (35bp) and a strong RBS (~10bp) upstream of the coding region, a terminator at the 3' end of your transcript (80bp), and cut sites that will allow you to insert the whole thing into a low-copy BioBrick plasmid (20bp flanking each side). First, let us work through the logic of planning the assembly:

Planning
First, step back and build exactly what you want your DNA to look like in your favorite DNA editor. I recommend using ApE for this. Make a list of things that your construction needs and include them in your DNA editor map of the gene. Now that you have in front of you what the finished construction will look like, you consider synthesizing the whole finished gene: the finished product will likely be in excess of 1300bp and so will likely cost >$1000. Your lab is running short on money, so you're not going to be able to afford so big a synthesis order. Since you can't order the whole thing, you realize that you'll have to put it together in parts. You also realize that since you're making a fusion protein, it will be difficult to use restriction endonucleases to assemble the two coding regions without inserting possibly deleterious AA residues between them. You therefore decide to attempt construction of the whole piece by assembly PCR, which will require template DNA for both pepA and gfp. You have DNA that codes for gfp on hand in a BioBrick vector. The DNA for pepA, however, doesn't exist and is too big to make by either primer annealing (200bp w/ IDT Ultramer oligo synthesis; this is very expensive, anyway) or primer extension (two 170bp ultramers will cost >$400; compared to a straight-up synthesis order, this seems too costly and risky, since the primers might just not work because of cryptic hairpinning or something weird). You decide to synthesize the 330bp of pepA with the 2xGGGS linker and RBS, which will run >$500, but since no other version of the DNA is easily attainable for PCR, you figure it will save time (and therefore money) if you just get it synthesized and sent in <2wks. Inclusion of the RBS and linker in the synthesized construct will shorten the primers needed to do PCR, which will enhance the chances of primers working as intended. So, now you have DNA for pepA and gfp and you want to assemble them by assembly PCR. You remember you need to include your promoter, terminator, and cut sites. All of these you can simply include in your PCR primers for the PCR reactions in the first step of assembly PCR. The primers will be big (>70bp) at times, but as long as there's a 20bp overlap w/ the template, the odds of the primers working are good. Also, if you have cut sites near the ends of your primers that you're intending to use, it's a good idea to include a 5bp spacer between the cut site and the end of the strand, since endonucleases sometimes loose efficiency if the cut sites are too close to the end of the strand (see the NEB catalogue appendix for how much room they need). With your primers designed and ordered and necessary templates in hand, you are now ready to begin building. Here is the set of steps necessary to complete construction:

Construction

 * 1) Do assembly PCR of your pieces. Perform the first PCR reactions (recommend: 50µl total volume of Invitrogen HiFi Platinum supermix) to amplify pepA and gfp with the necessary parts (promoter, terminator, cut sites) built into the primers.
 * 2) Run 10µl of the 50µl PCR reactions on a gel to confirm product.
 * 3) Use 0.5µl of each PCR reaction as template for the second round of assembly PCR. Use the leftmost and rightmost primers to amplify the assembled piece (residual primer from the previous PCR reaction are diluted to negligible levels).
 * 4) Run 10µl of the second PCR reaction on a gel to confirm product. Product should be the size of the sum of the two assembled genes.
 * 5) If you got product, purify your PCR reaction mixture (recommend: Qiagen's PCR purification kit).
 * 6) Cut your purified insert and target vector w/ necessary restriction endonucleases for insertion. Don't forget to heat-inactivate your enzymes (80*C for 20min usually does the trick) before ligation.
 * 7) Set up your ligation reaction.
 * 8) Do the transformation into competent cells of your choice (recommend: CCM80 Top 10 chemically competent cells).