Welcome back! Last time you performed PCR on the DNA pool you extracted from a gull sample. Specifically, you used primers designed to amplify bacterial 16S rRNA gene fragments from this complex pool. Now you want to systematically analyze individual fragments from this 16S DNA pool, which is aided by a process called cloning.
In traditional cloning, an insert is ligated to a backbone to create a circular piece of DNA called a plasmid. Typically, the insert and backbone are cut with special proteins called restriction enzymes (more about those in Module 2!) that yield complementary overhangs at the ends of the two pieces of DNA. The enzyme DNA ligase then chemically bonds the insert and backbone together by catalyzing formation of a phosphodiester bond. Ligated plasmids are selected in bacteria by including an antibiotic resistance marker on the backbone, and growing the bacteria in said antibiotic. (We’ll say more about that procedure below.) This method is especially suitable for cloning with the purpose of creating a well-defined piece of DNA.
Cloning overview: traditional approach.
An insert, such as a PCR product, and a backbone, such as a commercially available vector, are depicted in black and blue, respectively. These are digested with the same restriction enzymes (dubbed "X" and "E") so as to have complementary overhangs, and then usually gel-purified. Finally, backbone and insert are joined together to create a new vector using DNA ligase. Note: Figure modified from Prof. Bevin Engelward.
When diverse products must be cloned for subsequent analysis, as in our case, we would like to omit some steps from the above workflow. A few methods exist for cloning PCR products directly. The most popular approach is dubbed TA cloning, and it exploits the fact that polymerases such as Taq add an adenine to the 3’ end of PCR products. This single-base overhang alone can be used in a process very much like traditional cloning. Recall, however, that we used Pfu polymerase precisely for its precision, to improve our chance of successful amplification from a “difficult” sample such as stool. Pfu has proofreading capabilities, meaning that it doesn’t add spurious adenines.
Pieces of DNA that do not share complementary overhangs, that indeed have no overhangs at all but instead have blunt ends, can also be ligated together. There are several mechanisms by which this ligation may be accomplished. The simplest method and the one we will employ is the use of ligase. The enzyme must be highly efficient: blunt-ended DNA is not joined as easily as DNA with overhangs. An example of another method is so-called TOPO cloning, in which the biology of DNA topoisomerase I is exploited. Here the backbone includes two CCCTT sequences at the site where the PCR product will be inserted. Each sequence is cut by topoisomerase and remains bound to it; the two ends can ligate back to each other or to a newly introduced insert sequence. Whatever the method for ligation, the reaction mixture – in our case buffer, ligase, pCR-Blunt vector, and the PCR insert from M1D3 – is then added to bacteria.
Bacteria can take up foreign DNA in a process called transformation, during which a single plasmid enters a bacterium and, once inside, replicates and expresses the genes it encodes. Most bacteria do not exist in a transformation-ready state, but can be made permeable to foreign DNA -- by chemical or other means -- and are then termed competent. Competent cells are extremely fragile and should be handled gently, specifically kept cold and not vortexed. The transformation procedure is efficient enough for most lab purposes, with efficiencies as high as 109 transformed cells per microgram of DNA, but it is important to realize that even with high efficiency cells only 1 DNA molecule in about 10,000 is successfully transformed.
Blunt-end cloning vector. Image by Life Technologies.
After transformation, the bacteria are plated on an antibiotic-containing agar medium. In your case, the pCR-Blunt vector has a kanamycin resistance gene, and thus only bacteria that took up the vector can grow on kanamycin-containing plates, while untransformed cells will die before they can form a colony. Notice that a vector-containing bacterium will grow whether or not that vector contains a PCR product insert.
We would rather not waste our time analyzing colonies that contain pure backbone DNA! Thus, besides the KanR gene, pCR-Blunt contains a ccdB gene, which produces a protein that is toxic to E. coli; successful ligation of an insert interrupts the ccdB sequence, allowing bacteria containing this plasmid to survive. The ccdB gene is fused to lacZalpha and expressed under the Plac promoter, which can be turned on by the small molecule inducer IPTG, or is constitutively on in cells lacking lacI (the lac repressor gene). The One Shot TOP10 cells have just this genotype; how convenient! The cloning vector is shown with its key features at right.
So today you’ll ligate your 16S amplicons into the pCR-Blunt vector then transform the ligation pool, but only after visualizing that everything looks as expected on an agarose gel. Next time you will isolate DNA clones from individual colonies and send them off for sequencing. Then we’ll be just one step away from knowing what bacteria (a sampling of them, in any case!) are present in each gull sample.
Part 1: WRAP visit
Leslie will join us first thing today to talk about best practices for putting together a figure and its associated caption.
Part 2: Purify 16S PCR products
In many types of cloning, PCR products are purified prior to ligation. We are going to 'clean up' your PCR reactions prior to ligation to remove any extra components such as dNTPs and polymerase. Additionally, any very small off-target products will be removed.
- Transfer your complete PCR samples (2 bird samples + 1 NTC sample) to a 1.7 mL eppendorf tube and add 5 volumes of PB (e.g. if you moved 50 ul then 5 volumes would be 250 ul). PB is a high salt, low pH buffer added so the DNA in your reactions will bind the silica membrane. The salt in PB is guanidine hydrochloride, a chaotropic salt, meaning it will exclude water from the DNA, effectively precipitating it so it will bind the membrane in step 3.
- Get three QIAquick columns and collection tubes from the teaching faculty and label both the top and side of the tube with the appropriate reaction name. Pipet each PCR/PB sample onto the column. Microfuge the column in the collection tube for 60 seconds.
- Discard the flow-through in an empty 15 mL conical tube (label the conical tube "Qiagen Waste") and return the spin-column to the same collection tube. Add 750 ul of PE to the top of the column and spin as before. Qiagen sells PE and does not reveal all its contents, but the solution is at least 80% ethanol which keeps the DNA precipitated and on the membrane but washes the salt away.
- Discard the flow-through in your Qiagen Waste tube and return the spin-column to the collection tube once more. Spin for one more minute. Strange as it seems this is a very important step since it removes any residual ethanol from the membrane in the spin-column. If you forget this step the ethanol will elute with your DNA and interfere with loading your sample into the agarose gel. It may also inhibit the upcoming cloning step.
- Trim the caps off new eppendorf tubes and label the side with your team color and the date. Place the spin-column in the trimmed eppendorf and add 30 ul of nuclease-free water to the center of the membrane. Allow the column to sit at room temperature for one minute and then spin as before. The material that collects in the bottom of the eppendorf tube is your PCR product, ready to be loaded into a gel for electrophoresis.
Part 3: Gel electrophoresis of PCR products
You will use a 1.0% agarose gel to run your three PCRs from last time, as well as a reference lane of molecular weight markers (also called a DNA ladder).
- Add 4 μL of loading dye to each of three eppendorf tubes. No need to change tips in between.
- The loading dye contains bromophenol blue as a tracking dye to follow the progress of the electrophoresis (so you don’t run the smallest fragments off the end of your gel!) as well as Ficoll-400 (similar function as glycerol) to help the samples sink into the well.
- Now add 20 μL of each purified sample (your sample, your partner's sample, and the no template control) to individual eppendorf tubes and label them.
- Flick the eppendorf tubes to mix the contents, then quick spin them in the microfuge to bring the contents of the tubes to the bottom.
- Remember: to quick-spin, hold down the "short" button on your centrifuge for 3-5 seconds, then release.
- Load the gel according to the table below. Up to 4 groups will share each gel: 2 groups per lane.
- To load your samples, draw 20 μL into the tip of your P20. Lower the tip below the surface of the buffer and directly over the well. You risk puncturing the bottom of the well if you lower the tip too far into the well itself (puncturing well = bad!). Slowly expel your sample into the well. Do not release the pipet plunger until after you have removed the tip from the gel box (or you'll draw your sample back into the tip!).
- Once all the samples have been loaded, we will attach the gel box to the power supply and run at 110 V for 45 minutes.
- Later you will be shown how to photograph your gel. If your PCR gel shows that the 'no transcript control' sample contains an amplification product that is the same size as the 16S band or if your 16S samples contain multiple bands, alert the teaching faculty.
||Sample (20 μL)
||Sample (20 μL)
||DNA ladder (load 20 μL)
||DNA ladder (load 20 μL)
||Group 1, NTC
||Group 2, NTC
||Group 1, sample A
||Group 2, sample A
||Group 1, sample B
||Group 2, sample B
When all of the gels are loaded and running, we will have our pre-lab lecture for the day.
Part 4: Ligate purified product to backbone
- Prepare a clearly labeled eppendorf tube for each cloning reaction: yours and your partner's. Throughout the protocol below, perform the steps for each reaction.
- Our goal is to combine insert with 25 ng of vector with in a 10:1 molar ratio, using no more than 5 μL of insert.
- In the following order, combine: water for a final reaction volume of 10 μL; 2 μL reaction buffer; up to 5 μL PCR product insert; 1 μL of pCR-Blunt vector; 1 μL of ExpressLink T4 DNA ligase.
- Incubate at room temperature for 15 min. Meanwhile, obtain an aliquot of competent cells from the teaching faculty and let them slowly thaw on ice – this procedure takes about 5-10 min. Label the side of the tube (and also the top if you like).
- When both cells and reaction are ready, add 2 μL of the reaction to the cells. Do not pipet up-and-down more than once! Instead, gently mix the cells and DNA by twirling the tube in your fingers.
- Hold the tube near the top, so that the cells at the bottom stay cold.
- Incubate the mixture on ice for 30 min.
- Place the tubes in the 42 °C heat block for exactly 45 seconds, and transfer immediately immediately to ice for 2 min more.
- Add 250 μL of warm LB to each sample; do not pipet to mix.
- Place the tubes on the nutator in the 37 °C incubator, and rock them for about 1 hr.
- You may want to put a colored sticky label on the tube so you can quickly identify it later.
- During this incubation, you can complete Part 5.
- Shortly after starting your incubation, label and pre-warm two LB-KAN plates from the incubator (team/day/initials/sampleID/date).
- Sometime during this hour, get trained by your instructor on how to spread bacterial suspensions onto solid medium in petri plates.
Part 5: Prepare tubes for liquid O/N cultures
You will make your teaching faculty very happy if you contribute to their preparatory work.
- Please label 8 large glass test tubes with your team color and sample ID, per sample.
- Mix 25 mL LB with 25 μL of kanamycin.
- Using a serological pipet, aliquot 2.5 mL of LB+Kan per tube. These will be used to set up liquid overnight cultures from your 8 colonies for next time.
Part 6: Plate transformants
- Pick up an ethanol jar, spreader, lighter, and alcohol burner when you are almost ready to begin. Don't forget to wear your safety glasses!
- You will each have 2 plates so that you may plate your entire transformation reaction. Please add 150 μL of each transformation mix onto an LB-Kan plate. After dipping the glass spreader in the ethanol jar, you should pass it through the flame of the alcohol burner just long enough to ignite the ethanol. After letting the ethanol burn off, the spreader may still be very hot, and it is advisable to tap it gently on a portion of the agar plate without cells in order to equilibrate it with the agar (if it sizzles, it's way too hot). Once the plates are ready, place them in the 37°C incubator overnight; be sure that each is labeled with your team color, gull sample ID, and initials. One of the teaching faculty will remove them from the incubator and set up liquid cultures for you to use next time.
Due on M1D5
- You will continue to develop your abstract and data summary. For this assignment, you will prepare a figure and caption for the preparatory experiment in Module 1. This assignment should be submitted on Stellar.
- Begin by reviewing the assignment details.
- You should submit your PCR gel image as a well-labeled figure with an appropriate figure caption. Be sure to follow our written guidelines as well as the suggestions presented by your writing instructor during class.
- Below the figure, interpret the outcomes in concise language. Consider reviewing slides 30 and 31 in the Poster Best Practices Talk from the BE Communications Lab to learn how to turn bulky sentences into succinct bullets. It's also okay to use complete sentences when they more clearly make your point.
- Please see this example for a suggested layout. For this assignment, you do not need to turn in a draft of the background/motivation section. But use the Data portion as an example of an acceptable layout.
- Select an article for your Journal Club presentation. Please see the M1D9 page for the lists of suggested articles and assignment details.
- NEB Loading Dye (link)
- 2.5% Ficoll®-400
- 11mM EDTA
- 3.3mM Tris-HCl
- 0.017% SDS
- 0.015% bromophenol blue
- pH 8.0@25°C
- 1.0% agarose gel in 1X TAE
- 1X TAE
- 40 mM Tris
- 20 mM Acetic Acid
- 1 mM EDTA, pH 8.3
- Zero Blunt PCR Cloning kit from Life Technologies
- Includes One Shot TOP10 chemically competent cells
- Luria-Bertani broth from Teknova
- 1% Tryptone
- 0.5 % Yeast extract
- 1 % NaCl
- Qiagen QIAquick PCR purification kit
- PB reagent with Guanidine HCl
- PE reagent with Ethanol
- EB reagent = 10 mM Tris, pH 8.5
- Kanamycin: 25 mg/mL, aqueous, sterile-filtered
- LB+KAN plates
- LB with 2% agar and 25 μg/ml kanamycin
Next Day: DNA sequencing
Previous Day: 16S PCR and paper discussion