Lecture notes for Molecular Biology and Techniques

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0.0 LECTURE NOTES SECTION DAY OF COVERAGE MOLECULAR TECHNIQUES LECTURE NOTES 2013 BY DR. DAVID NG ADVANCED MOLECULAR BIOLOGY LABORATORY MICHAEL SMITH LABORATORIES UNIVERSITY OF BRITISH COLUMBIA HTTP://BIOTEACH.UBC.CA HTTP://SCQ.UBC.CA HTTP://WWW.MSL.UBC.CA SUBMIT TO OUR SCIENCE WRITING PROGRAMS. 0.1 CONTENTS: BY SECTION BY LECTURE DAY 1.1 DNA: General Info 1.2 DNA: General Workflow Before the start of workshop, please read APPENDIX A (on replication) 2.1 DNA: Genomic/Plasmid 2.2 DNA: Cell Lysis 2.3A DNA: Nucleic Acid Purification MONDAY 2.3B DNA: Nucleic Acid Purification 1.1, 1.2 2.4 DNA: Precipitation 8.1, 8.2, 8.3, APPENDIX B 2.5 DNA: Quantitation 4.1A, 4.1B, 2.6 2.6 DNA: Agarose Gel Electrophoresis TUESDAY 3.1 CLONING: What is a Vector? 4.1A, 4.1B, 2.6 3.2 CLONING: Choosing a Vector I 2.1, 2.2, 2.3B, 2.5 3.3 CLONING: Choosing a Vector II 3.1, 3.2, 3.3 3.4 CLONING: Plasmid Preps 4.2, 5.1, 6.4 4.1A ENZYMES: RE Digests I WEDNESDAY 4.1B ENZYMES: RE Digests II 7.1, 7.2, 7.3 4.2 ENZYMES: Ligases/Phosphatases 2.3A, 2.4 6.1, 6.2 5.1 TRANSFORMATION THURSDAY 6.1 PROTEINS: General Info 3.4 6.2 PROTEINS: SDS PAGE 2.3B 6.3 PROTEINS: Western Blot 6.4 6.4 PROTEINS: 2D Gel Electrophoresis 8.4 7.1 RNA: General Info 7.2 RNA: Isolation and Purification FRIDAY 7.3 RNA: Gene Expression 6.3 8.1 PCR: The Basics 8.2 PCR: The Specifics 8.3 PCR: Troubleshooting 8.4 REAL TIME PCR APPENDIX A: Replication APPENDIX B: Hybridation/Stringency 1.1 DNA: General Info. DAY 1 First off, some basic things to consider that ultimately affect how you envision the behaviour of DNA in your experiments: 1. DNA is a simple molecule No really it’s got your A, T, C and G, which from the eyes of a chemist, are pretty similar anyway. What does this mean? It means “predictability” which is a very handy thing in research. 2. DNA is negatively charged Very much so – lots of electrons whizzing around those oxygen groups. 3. DNA has lots of ring structures You remember things like pi orbitals, and the difference between polar and non-polar bonds? Anyway, the point is that ring structures are generally quite hydrophobic. 4. DNA is a very robust molecule. Spat in your sample? Probably o.k. Left out at room temperature over the weekend? Not a problem. Generally, the easiest of the biological macromolecules to work with). 5. DNA work has lots of toys. (Talk about accessories You simply have a lot of options as far as what you can do with this molecule, whether it is a procedure, or a specific enzyme, etc) Some general common sense rules that are worth applying to all DNA experiments, and frankly, while we’re at it, to all molecular experiments. 1. Know the experiment’s level of forgiveness. Another way of saying that it pays to know the chemistry of your procedure. Inevitably, each experiment has a degree of forgiveness, which is a really useful thing to know. This allows you to gauge your level of care, which in turn will reflect on your efficiency as well as your ability to troubleshoot. 2. Get your sample as pure as you can. Although this is related to point one, consider the following thought: any impurity is an outright invitation for caveats. i) If you have contamination, your subsequent steps may be hindered, or worse, unduly affected. ii) If you have a contamination, you might just lose your sample outright. i.e. DNA doesn’t like nucleases. And nucleases are “everywhere.” 3. If you had to choose, be gentle rather that be rough. Doesn’t hurt to be careful when handling material. i.e. keep everything cold, since these enzymes are much more active at physiological temperatures. (i.e. use of "ice cold" this and that") Wear gloves, etc. 4. Know the idiosyncrasies of your molecule: At times, you need to be aware of specific nuances that apply to your particular “brand” of molecule. For instances, genomic DNA is different from plasmid DNA is different from a PCR product is different from an EST fragment. 5. Think carefully about how much stuff you actually need. Small amounts generally easier to work with, machines are smaller, take less time to operate, etc, etc, etc… So, if you know, you don’t need much, then only use that amount. 1.2 DNA: General Workflow. DAY 1 In general, when doing molecular work with DNA sequences, there are a number of basic things you’re trying to do. Often, instructional manuals will attempt to classify this ilst as a “general workflow” which is akin to a sort of flowchart of events. In a nutshell, this tends to involve the following: 1. Figure out what it is you’re interested in: This step tends to be based on literature search, or via an observation that one has deemed intriguing. Basically, this narrows the boundaries of the DNA code you’re keen to explore. A defining feature of this step is also figuring out where this sequence might come from, as well as its likely size. Both of these parameters will greatly influence subsequent steps. 2. Physically retrieve the sequence that you are interested in: Once you know what you’re interested in, next comes the actual work to get it. This might involve working with tissue/cells which tends to incorporate various cell lysis and nucleic acid purification procedures. Or it may entail working with material that is provided in purified form as a pre-determine lab sample. As well, there are often steps to modify this DNA sample so that it can be used for subsequent steps,: this might include finessing its size, or simply amplifying it to appropriate amounts. Note that the most common way of performing all of these steps often involves the Polymerase Chain Reaction method. 3. Insert your sequence of interest into an appropriate vector. Although this will be described more fully, harbouring your DNA of interest in a specific vector will define how you intend to “characterize” your sequence of interest. i.e. Do you want to sequence it? Do you want to transcribe/translate into a protein? Do you want to insert your sequence into a host cell for observation? etc. In all, this workshop will cover a wide variety of different techniques that provides a myriad of different options for you to cover this “general workflow.” Example Workflow GET TISSUE LYSE CELLS PURIFY GENOMIC DNA PRECIPITATE TO CONCENTRATE PCR DESIRED FRAGMENT CHECK ON AGAROSE GEL PURIFY FRAGMENT / QUANTITATE LIGATE INTO VECTOR SEQUENCE VECTOR (TO CHECK FRAGMENT) TRANSFORM/TRANSFECT INTO HOST 2.1 DNA: Genomic/Plasmid DAY 2 We’ll talk later about plasmids, so will focus primarily on genomic DNA. Genomic DNA? What is it? Well, it’s actually trying to retrieve “all” of the sequence found within an organism. Wiki also says it succinctly: “In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA.” From a practical point of view, if you’re working with bacterial or eukaryotic samples, you are trying to isolate all DNA from your sample. By the way, many species of genomic DNA samples involve big, complicated pieces that often end up looking like snot in solution. Why are you getting genomic DNA? (a couple of reasons) 1. Profiling/Fingerprint: This is the stuff you use to compare blood/semen samples. Essentially, your genomic DNA can lead to some form of reproducible data that provides a unique representation of the sample (i.e. band profile on a PCR DNA fingerprint for example) 2. First attempt at cloning a new gene. If you plan on looking for something completely new and novel. Therefore, you will need to go to the original source of that code which is the genome. And although, you may be able to get protein or mRNA information before this step, you will ultimately need to define it within a genomic context (i.e. what chromosome is it on, promoter regions, intron/exon organization, etc etc.) 3. Characterization/Diagnotics. Lots of examples in basic research. I.e. you’ve made a transgenic/knockout organism, and you need to check if indeed, the modification has occurred – this starts with a genomic prep. You have an interesting phenotype and you want to look deeper. i.e. a disease state. This hunt may often start from a genomic prep. 4. Studying genome for clues into gene expression and regulation: Although, usually the cDNA for a gene gets a lot of attention (this is a DNA copy of the messenger RNA directly responsible for coding the protein), this edited form of the gene misses out a lot of stuff that may be interesting or relevant to your research. I.E. Study gene expression regulation, euchromatin nuances, tissue/developmental specificity, spliced isoforms, etc..... 2.2 DNA: Cell Lysis. DAY 2 IT'S IMPORTANT TO REALIZE that there are many variations of the lysis procedure. Some are quicker, some are more efficient, some are more expensive, some only work in certain situations. In our PCR experiment, we performed a simple boiling step to get the job done. If there’s time in class, we’ll also mention the SDS/proteinase K method. It being a very standard procedure (often embedded in kits). NOTE: Large pieces of Genomic DNA can shear. (not actually a big deal for most procedures but why take the risk). In this case, dealing with a piece of DNA that is 100kb + in size. No vortexing, no vigorous pipetting/snip the end of your tip before use. EXAMPLE OF COMMON LYSIS PROCEDURE: (SDS/Proteinase K): 1. Place the cells in a “physiological” like buffer. There are many varants of this, but the below is a good example. Tris (buffering agent - good pH6-8) Often at the 10 – 20mM range. EDTA chelates divalent cations which are necessary cofactors for DNase activity (way of shutting down nucleases) Usually at the 10mM range. NaCl at physiological concentration (generally considered to be 100 – 150mM) Keeps all molecules happy (particularily proteinase k)/prevents unwanted aggregation. NOTE that TE buffer is a commonly used buffer which contains the aforementioned Tris and EDTA. 2. Then add the reagents which are responsible for the lysis. SDS, nasty ionic detergent/ good at breaking membrane, general denaturant (inhibit enzyme activity). Since DNA is so robust, not really adversely affected by SDS treatment. Note: When dealing with plant material, a very common detergent in place of SDS is Sarkosyl. This essentially behaves in a similar manner to SDS. Proteinase K-serine protease (works well at 55C), used because it is very effective and not particularily susceptible to SDS, and other denaturants such as urea. Proteinase K will chew up protein, which helps lysis in general and frees up the DNA from any protein gunk associated with it (euchromatin structures/histones, etc). Best used FRESH (quite an important step). Incubation step generally a minimum of an hour. Although most will allow the procedure to go longer (this may be largely dependant on material being used – i.e. many procedures outline an overnight or 16hrs incubation time). I.E. mouse tails - may want to go overnight). 2.3A DNA: Nucleic Acid Purification. DAY 3 There are a number of different ways to purify nucleic acids (indeed, with our PCR experiment we are just using special CHELEX beads which are designed to pull out positively charged ions). For the remainder of the course, we’ll be utilizing three other methodologies described below. A. DIY METHODS PHENOL/CHLOROFORM NOTE that Phenol is pretty nasty It will burn. (Apparently, someone once told me that if you cover 5% of your body, you will die). Since we are using reasonable amounts, do PHENOL addition in fumehood, but all manipulations can occur at the nech. If you get it on you - not panic mode. Quickly rinse off with cold water. Phenol, by the way, is usually buffer saturated. (i.e. when you buy it or make it, the solution comes in two layers - top layer is excess buffer, and bottom layer is buffer saturated phenol. This is because the pH is important - In order for our purification to work, we need the PHENOL at a neutral pH.( acidic pH is not good as DNA becomes soluble in phenol). The fact that it is saturated is also important because this means that any additional aqueous (i.e. water) solution you add will create it’s own fluid layer. This purification procedure works on the principle of "differential solubility". 1. To your lysate, you will add an equivalent volume Phenol/Chloroform/Isoamyl alcohol. (usually at a volume ratio of 24:23:1). Phenol – organic solvent/ nucleic acids not soluble at all. Therefore, DNA/RNA will stay dissolved in aq phase. Lipids and polysaccharides preferentially go into the phenol phase. Proteins will also selectively go into phenol solution. 2. FURTHERMORE phenol also acts as a denaturant, proteins denature form aggregates and will collect at the interface. You will see GUNK the interface. 3. Choloroform, also has same general attributes as phenol (as far as solvent properties) but also stabilizes the rather unstable boundary between aq and organic layers. Isoamyl alcohol also contributes to interface stability and also helps prevent frothing. Generally you do this step 2 or 3 times. The more times you do it, the cleaner your sample (you may even note that the interphase gets cleaner and cleaner with each step). Note this procedure is very reliable and does not lose much DNA yield. This is probably why a lot of labs still like to use it. (BACK EXTRACT: adding extra aqueous to your organic samples). Sometimes do a final Choloform step. Here, the interface is a little trickier to handle. But this step is a good safeguard to prevent any organic carrying over to your final aqueous solution. Likely finish with a chloroform step because it evaporates easily (?). In other words, this give you the option of leaving the lid open to really make sure ALL your organics are gone from your prep. 2.3B DNA: Nucleic Acid Purification. DAY 2/DAY 4 B. KIT BASED METHODS pI BASED KITS We’ll first be checking out Invitrogen’s ChargeSwitch kits, which rely on magnetic beads that contain polymers with neutral pI. Basically, if the environment is such that the net charge of the beads can bind to the DNA, you can release the DNA by altering the pH environment of the solution in the other direction (i.e. change the charge of the polymer). In short: If pH pI (more acidic), then the net charge of polymer linker becomes more positive (higher affinity for DNA) If pH pI (more basic), then the net charge of polymer linker becomes more negative (lower affinity for DNA) Fast But you would need to invest in special magnet racks. SILICA BEAD BASED KITS (i.e. QIAprep) Nucleic acid purification kits more commonly involves the use of silica based beads which are specifically designed to interact with the very electrostatically charged nucleic acid molecules. Our QIAGEN preps are a good example of how kits work in general: 1. Our DNA sample was first treated with a salt solution (something like NaI). This stuff is usually classed as a chaotropic salt, which is really a fancy way of calling a salt that is capable of altering structures by interacting with and thereby sequestering many molecules of water. In other words, salts like NaI can bind to several molecules of water at high stoichiometry, depriving water from the DNA structure in particular. This will alter its shape/charge/etc which makes it specifically bind to the silica beads (often pretreated themselves with ions to make them more amenable to specific binding). 2. We next do a wash, which in our case is called NEW WASH buffer. Not entirely sure what this is, but you can bet it doesn't affect the electrostatic state of your nucleic acid (likely an alcohol/water mixture). You don't want 100% water during the wash steps, or else your DNA will get the water back and fall off the beads 3. Which is why the final elution steps are water alone. THERE are lots of variations of these kits. Popular these days are versions that are set up in a column format. Arguably, most popular are those sold by Qiagen. 2.4 DNA: Precipitation. DAY 3 PRECIPITATION OF DNA (alcohol and salt procedure) To precipitate nucelic acids, you can add a high concentration of salt (i.e. Sodium Chloride or Ammonium Acetate). Why? Helps in the precipitation of the DNA in EtOH. NOTE: sometimes, you can also omit salt entirely (dependant on concentration of DNA). Salt will help neutralize negative charge of DNA (will also sequester the solvent molecules - in this case water) Salt will also interact with water, thereby weakening it’s solvating prowess. This is commonly known as “salting out” Use 100% EtOH. Time frame (show graph) EtOH generally helps because it is a much crappier solvent than something like water (which is very polar). DNA will tend to stay precipitated in 65% Ethanol. NOTE that efficiency of EtOH precipitation is dependant on a number of things. Temp, time, amount of DNA. Can also use isopropanol for precipitation steps. RNA tends to stay soluble in this solvent (selective precipitation). which is why some people use it for this purpose. DNA will tend to stay precipitated in 50% isopropanol. Once precipitated, DNA will tend to stay insoluble if kept in 70% ethanol. Hence, a lot of washing steps tend involve Ethanol based solutions, which is important as it removes any excess salt present in your sample. Final resuspension of sample in TE. Graph below highlights how, generally speaking, unless working with very small amounts, DNA precipitates pretty quickly. 2.5 DNA: Quantitation DAY 2 Spectrophotometry readings: Using UV absorbance to ascertain DNA/RNA amounts and purity. Ring structures can absorb UV wavelengths. BUT Lots of things have ring-structures (including nucleic acids, proteins, organics, detergents, lipids, the list goes on and on...) i.e. Both DNA and RNA - ring structure absorbs strongly at 260nm and 280nm. Protein = some amino acids like W and P and Y also flouresce strongly at 260nm and 280nm. Bottom line is that you have to take numbers with a grain of salt. Very rough estimate, since lots of things absorb at UV wavelengths. Also quite sensitive to pH which is why dilutions are often done with TE buffer at a particular pH, that was also used to calibrate the spectrophotometer. You can also assess purity by looking at ratios of 260 over 280. DNA A260/A280 1.8 RNA A260/A280 2.0 These are good ratios for purified product. If you get good numbers here, then maybe you can also get good quantitation numbers (NOTE: that the conversion for O.D. numbers to DNA amounts is the constant 50ug/ml per O.D260nm value, LIKEWISE, the constant for RNA is 40ug/ml per O.D.260nm value.) Other wavelengths of interest include: A230 (good especially when phenol is in the mix, since phenol absorbs most strongly at 270nm). Quantitation Assays: Usually rely on stoichiometry binding between reagents like Ethidium Bromide, SYBR dyes to correlate with a DNA amount. Generally a more robust, more expensive manner of determining amounts. ALONG the same lines, DNA fragment quantitation can be done by comparing band intensities in an ethidium bromide stained gel – i.e. use a marker as a gauge. 2.6 DNA: Agarose Gel Electrophoresis. DAY 1/2 Using Agarose. Polysaccharide polymer. Used because of its ability to form pore sizes capable of resolving 0.2kb to 60kb, (200bp to 60,000bp). Essentially, creates a big mesh of fibers that your DNA has to pass through. Electrical charge is the driving force and things will separate according to size. DNA works well in this set-up because for things to separate in correlative manner, all DNA species generally have about the same charge density. ALSO, in our case, the DNA we are looking at has been cut with restriction enzymes - therefore all DNA fragments are predominantly linear in shape. Loading buffer: Glycerol: thickens sample up so that it doesn’t float away after you load it into the well 0.1 M EDTA stop reagent for the assay 1% SDS help denature the RE stop the reaction 0.1% bromophenol blue. Dye. Helps you visualize sample when loading. Will run towards +electrode. Can use it as a rough idea of where your DNA may be running (dependant on gel%) Running buffer: Tris Borate EDTA (TBE) In this case, borate is your ion, which allows the generation of an electric field in the gel set-up. Your common alternative is TAE: Here, acetate acts as an alternative ion - is often used because it works and is much cheaper. Need 50c for 10L of TAE, (need 10 for 10L of TBE) But tris/borate has a significantly better buffering capacity, which means gel running is more reliable especially at high voltages (for speed), or long running times (i.e. overnight). BUT borate (when preparing the gel in microwave or oven) also forms complexes with the agarose sugar monomers/polymers. Can be a problem if using procedures to isolate band from a gel (i.e. melting of gel is required), although most band extraction kits come with chemistry to deal with this. Quick mention of Sodium Borate systems (cheaper, very fast, but resolution can be affected – Kern and Brody, BioTechniques Feb 2004) Visualizing the DNA: Most people now use SYBR stains Examples include SYBR SAFE,/SYBR Green I/ SYBR GOLD which are exceptionally sensitive nucleic acid gel stains with bright fluorescence when bound to dsDNA and low background in gels, making it ideal for detecting dsDNA in gels using laser scanners or standard UV transilluminators. Compared to Ethidium Bromide, these stains are generally about a hundred fold more sensitive, and less carcinogenic to boot (a bit more expensive mind you, and you will need a particular filter in your light box to see it – although this isn’t that expensive). Stain can occur during run, or after run. Alternatively, you also have Ethidium Bromide (carcinogen), also sensitive stain that interchelates DNA (which has the added ability of slightly uncoiling it). Need to use a UV lightbox to see it – take care to use appropriate eye shielding. Some labs add EtBr into gel. Some add it after gel has run (i.e. stain with solution containing EtBr) - adding it into the gel is much easier, but if the apparent molecular weight of closed circular DNA is particularily important to you, it may be worth adding after so that it doesn’t affect its molecular weight. FOR EtBr, you should inquire at your health, safety department, as many research facilities are phasing out EtBr use. 3.1 CLONING: What is a Vector? DAY 2 Vectors are essentially shuttles that carry your DNA. At the most basic level, almost all vectors have the following traits (sorry for the long long titles): UPKEEP: Naked DNA (no matter how important it is to your research) does not upkeep itself. Therefore, to do this, you need your “DNA of interest” to replicate independently of integration into host genome. i.e. the vector allows the use existing replication machinery of host organism. OR another way to get around this, is for the vector to contain information that can drive integration into the host genome. PLACE TO PUT DNA IN: all vectors generally have defined places where you can insert your DNA of interest. This is usually at a place called the Multiple Cloning Site (MCS), and is governed by (i) convenience (i.e. easy to put something in this area), or (ii) geography (you put your DNA “here” because it is next to some element – say a promoter – that you want to use to act on your DNA of interest.” Vector nomenclature, while hardly poetic, is usually a reflection of what the MCS and it’s surrounding geography is all about. SOMETHING THAT LETS YOU KNOW IF THE VECTOR IS IN THE ORGANISM: All vectors will generally contain a selectable marker (notable exception is where GM crops are involved). This is usually a gene that confers resistance to some sort of drug. Commons ones used in research field include antibiotics such as ampicillin (common for e.coli), and neomycin (common in mammalian cell cultures). On the other hand, some organisms will rely on auxotrophic traits. This turns up a lot in yeast work, whereby host cells are mutant and unable to make a nutrient like tryptophan. Sooo... In order for these strains to grow, you either need to supply it in media, or you insert a vector into the yeast that reconstitutes the ability to make trp.. SOMETHING THAT LETS YOU KNOW IF THE DNA OF INTEREST YOU PUT IN IS ACTUALLY IN THE VECTOR, WHICH IN TURN IS IN THE ORGANISM: There are a number of strategies that let you determine whether your DNA of interest actually made it into the vector. These strategies often entail some form of insertional inactivation, or ligation trick (i.e. CIP assay, T overhang systems). 3.2 CLONING: Choosing a vector I DAY 2 There are two general things to think about. First of all is the principle that size matters. i.e. How big is my DNA of interest? Is it a relatively small 10,000bp cDNA, or is it a chunk of chromosome 300,000bp? In this respect, here is a run down of common vector types: 1 PLASMID VECTORS: such as pUC18. (used in our experiment): a) selectable marker: ampicillin resistance cassette. Therefore if you want to ensure that your bug has gotten the plasmid, then you just test for the bug’s ability to survive under drug treatment. b) Blue white screening: which sort of works like this - Things to point out -LacZ gene (b- galactosidase) Bugs will constitutively express a lac repressor. Which interacts with lacUV promoter. Therefore, no transcription of beta-galactosidase. But if you add lactose - lac repressors fall off promoter. What you usually use is IPTG (isopropyl-b-D-thiogalactoside) which is an analog of lactose. Anyways, if there is NO insert - lacZ gene will get transcribed. Codes for a b-galactosidase. If there is an insert - something else will get made. Frameshift mutations/insertional inactivation. Will not get lacZ transcribed. c) MCS: Small area of the plasmid where the vector designers have decided to put lots of unique restriction sites in a small area. I.e. try to make it as easy as possible for you to have a convenient nice restriction site that works for your cloning strategy. 2 BACTERIOPHAGE VECTORS: (very old school – but will cover as background necessary): fancy word for virus that infects bacteria. Good for moving around 20kb worth of DNA. Good example of bacteriophage vector is the "lambda DNA." This thing is about 49kb in size, containing essential and non-essential regions as outlined in figure. Essentially, the key idea is to take advantage of the viral pathways to get your DNA inside. I.E. you can package your DNA into a bacteriophage structure, which can infect and shunt your DNA into the host cell (a bug). ONCE the DNA is inside, the bacteriophage will still go about making more DNA, and making all the necessary components to make more of itself. This is essentially how you upkeep your DNA. The only technical difficulty is how you would "harvest material" . i.e. your DNA is not in the bacteria per se, but rather is inside the bacteriophage, and the logistics of getting bacteriophage cultures are more difficult (i.e. plaques on a plate)... I.E. WHY phage systems are now rarely used. The other reason is because of... 3. COSMID VECTORS: Good for packaging 30-45kb of material. Think of it as an unworthy bacteriophage. I.e. Even essential regions have been deleted (this is why it can carry more than normal bacteriophage vectors) but special cohesive sites (COS SITES) are still present. Therefore, still has enough genetic information to get packaged but not enough to "be a bacteriophage" i.e. doesn’t have information to make more bacteriophage. NOW you can use a KIT to supply materials (bacteriophage bits and packing machinary) needed for packaging of your DNA into a "pseudo" bacteriophage. Then your bacteriophage can infect a host organism, and effectively transform it (deliver your DNA). ONCE, DNA gets in, the COS sites will direct recircularization and you now essentially have a BIG PLASMID (therefore can use normal plasmid techniques for isolation). 4. ARTIFICIAL CHROMOSOME SYSTEMS – BACs and YACs: "The realization that the components of a eukaryotic chromosome that are required for stable replication and replication in YEAST, are VERY SMALL very DEFINED sequences” CAN TAKE UP TO 1 Mb NOTE that BACs or Bacterial Articifical Chromosomes (sometimes known as PACs or plasmid artificial chromosome) are more common nowadays. Similar to YACs in principle in that they allow incorporation of a huge amount of DNA (up to 300kb). The difference is that you can work in e. coli which is much easier than yeast manipulation 3.3 CLONING: Choosing a vector II DAY 2 The other constraint, of course, is what you want to do with your DNA of interest, and quite literally, to say that the world is your oyster is very much a reality. Here are two examples of the sorts of things you need to think about. 3.4 CLONING: Plasmid Preps. DAY 4 Isolating plasmid DNA from other types of DNA (i.e. genomic) is actually very simple. In short, it usually involves a denaturing step, followed by a quick renaturing step. The IDEA is that plasmid DNA being much smaller, can renature relatively easily - consequently, once back to normal it can go into solution easily. Something like genomic DNA will have an incredibly hard time renaturing because it is simply too big and too complicated. It doesn't renature effectively and instead tangles up and precipitates out. IF YOU THINK ABOUT IT, you have now separated your plasmid (in solution) from your genomic (out of solution) prep. You simply have to centrifuge away the genomic pellet, and you are left with your plasmid DNA (+ all the other cellular crap like proteins, etc etc however, now you can use any standard DNA purification procedure). Most common way of doing this is known as the ALKALINE LYSIS METHOD (which also makes an appearance in practically all kit based plasmid prep methodologies) Here the idea is to chemically denature and renature. 1. Need to open the cells up. NaOH and SDS. Ruptures cells, and denatures everything. Low pH specifically breaks H bonds in dsDNA. O.K. so your test tube is now this messy mix of denatured stuff. Genomic DNA (big) - denatured. Plasmid DNA (small) - denatured. Proteins - denatured. THROW IN salt that is acidic (KAc pH4.8). Salt helps in the precipitation process. Acid - causes things to go back to neutral. DNA can renature BUT HAPPENS VERY QUICKLY. Large DNA renatures as a MESS. Small DNA renatures O.K. So, genomic DNA will precipitate out (should see a white mess), but your plasmid DNA will now be in solution. TA DA Move onto purification/precipitation step. ALTERNATIVE QUICK AND DIRTY PLASMID PREP METHOD: Via causing the cells to lyse by using STET + lysozyme. and then the trick is to boil and then cool (this provides the denature and renature step) 4.1A ENZYMES: RE digests I READ ON YOUR OWN Generally speaking, molecular biology enzymes are like your high maintenance buddies. It really pays to know them well, and they will end up being very useful to you. We will focus on a lot of details with regards to Restriction Endonucleases (REs), so that you’re aware of the sorts of nuances entailed. NOTE: Enzymes are pretty expensive so you want to keep them cold and keep them clean. This means you always use a freezer box when keeping them at your bench. This means that if you don’t have a freezer box, you do everything at the freezer This means no double dipping. NOTE: bug a vendor and get some free catalogs – these are very useful reference source. Especially useful is the New England Biolabs catalog. 1. WHAT IS A RE?: Think of it as an enzyme that cuts or degrades DNA but in a very specific manner. There are actually several classes of REs, but the most commonly used are affectionately categorized by the type of cut they leave. In shop talk, the two common types are referred to as “sticky end” and “blunt end” cuts. In terms of physiological or historical background: All started in 1970. Hamilton Smith at John Hopkins University studying Hemophilus influenza. - extracts from this bug could cleave DNA at very precise points. Turns out, bugs have sets of restriction enzymes which are believed to serve as their "immune system" of sorts protecting them from virus infection (viral DNA gets cleaved). To date well over 3000 diff REs to choose from, which were found from screening 10000 bacteria. 2. WHY ARE YOU USING A RE?: Generally speaking, you are trying to create a specific cut, often to obtain a particular fragment or chunk of DNA. However, RE can be used to simply “alter” the size of a DNA sample to fulfill a particular purpose – a good example is to use a RE to cut genomic DNA down to size for effective agarose gel viewing. In some respects, this ties into the idea of WHICH ONE TO USE? 3. HOW MUCH TO USE?: Amount? Very simple. 1 unit of enzyme is enough (theorectically) to do whatever it’s suppose to do (in this case “cut”) 1ug DNA in a specific amount of time at the defined temperature (in a reaction volume 50µl) BUT it’s interesting to point out that every enzyme can come with its own personality. (which, in turn, can be dependant on the batch or company they come from). Common restriction enzymes generally work quite well, and arguably, any enzyme that is not “fancy” is more reliable. NOTE that generally speaking blunt cutters seem to be more finicky than sticky cutters. ALSO NOTE that researchers often add more enzyme than is theorectically needed (likely to ensure that the reaction will go to completion, but probably it’s simply impossible to pipette anything less than 1.0µl accurately anyways). 4.1B ENZYMES: RE digests II READ ON YOUR OWN 4. CONDITIONS OF THE REACTIONS: enzymes will always come with "special" buffer. Generally, the buffer is all about making your enzyme happy - keeps it at a particular pH (usually around 7 to 8), a particular salt concentration, and has cofactors like Mg in it. Salt is the most varied consideration in getting a reaction to work (In fact, in the not so recent past, buffers use to be separated simply as low, med and high salt.). This is particularily true if you wanted to cut your DNA with 2 different REs (double digest). In the past, you would cut first with RE that worked at lower salt / bring up the salt for second enzyme. (this would be an example of a tough dilution question) Nowadays, there are handy dandy buffer activity charts – lists % activity for an enzyme under different buffers. 5. OTHER THINGS YOU CAN ADD. RnaseI – get rid of extra RNA in your prep. BSA – (albumen) generic protein. Proteins have an uncanny ability to destabilize when solvated in a VERY dilute concentration (why? Too much water solvating protein) Companies try to fix this by adding more (generic) protein in solution so that overall protein is higher. Spermidine ( not used as much anymore): Added b/c of its ability to interact with polymerases and other DNA binding proteins. DTT: reducing agent. Dithiothrietol - during purification process by company - May get unwanted disulfide bond formation, which would affect enzyme structure. 6. TIME AND TEMPERATURE: only guidelines. In terms of RE, the unit value is usually under time constraint of 1 hour. Therefore, 1 hour is the bare minimum. However, it is considered safe to leave digests going overnight, which is often suggested if it is crucial for you to get complete digest. Some enzymes sensitive to high temperatures: useful if you want to inactivate it (heat inactivation). 7. QUALITY OF YOUR DNA SAMPLE : As a whole, restriction digests are very forgiving. BUT no organics (i.e. phenol or chloroform), as they will affect RE viability. METHYLATION: will also affect the ability to be cleaved. IN actual fact, this is what protects the bacteria from its own restriction enzyme defence system. I.e. it has a corresponding methylation enzyme that protects bacterial DNA. This is why most e. coli strains that you work with have dam- and dcm- nomenclature. This means that these methylation enzymes have been knocked out so that they won’t affect the ability to cut DNA isolated from these bacteria. - important point since all organisms capable of methylation to some degree. 7. STAR ACTIVITY: This is when your RE gets sloppy, loses specificity. The point being to remember that these are enzymes, and to think of the sequence specificity as its substrate. Things that can cause non-specific cleaving include things that can affect the enzyme-substrate relationship: (i) beyond saturating kinetics: too much of anything (ii) change active site: buffer conditions, contaminants. 8. STOPPING REACTION: Easy. +EDTA or heat the sucker, as some enzymes are heat sensitive. (Stick in a 65C waterbath for 10min). When in doubt, purify the DNA out, by doing something like the phenol chloroform. 4.2 ENZYMES: Ligases/Phosphatases DAY 2 LIGATION REACTION: Ligase: enzyme that can anneal two pieces of compatible ends of DNA together. The important nuance to consider is that the 5’ end of your DNA ends need to be phosphorylated. From a compatibility point of view, if you are dealing with sticky ends (i.e. they have the overhangs), the overhangs MUST be complementary to each other. Blunt ends can be useful in that all blunt ends are compatible with each other. NOTE that ligation works way better for sticky ends then for blunt ends. T4 Ligase is considered the workhorse for this particular procedure. Conditions vary greatly depending on the one you use, and where you bought it from, but nowadays, the procedure can be done at room temperature in as little as 15 to 60 minutes. can’t directly check if ligation worked except by virtue of getting bugs to grow after transformation. -useful to try different insert:vector ratios. Molar ratios that often tried 1:1, 3:1, 5:1 etc. Generally more insert is better, but every ligation is different. CALF INTESTINAL PHOSPHATASE (CIP) Can also use alkaline phosphatase: Used to dephosphorylate 5’ phosphates. Useful to desphorylate a cut vector, which prevents recircularization of vector unless an insert can get in and provide the 5’ phosphases. (i.e. it's a way to make sure that any colonies you see on the ligation plates MUST have an insert in the multiple cloning site) 5.1 TRANSFORMATION: DNA into your host. DAY 2 The story so far… You have just done a ligation reaction where you have added a cut vector and a fragment together. -You want to know if the ligation worked… FIRST: you are going to put the ligation mix into bacteria, and plate the bacteria onto ampicillin supplemented media. Therefore, in order for bugs to grow - MUST have ampR. Therefore must have plasmid. But plasmid must be circularized in order for it to get continuously and independantly replicated. Therefore, LIGATION HAS to work to get all of this. Is the insert in? Use the white blue system (your plates will also have X-GAL) TRANSFORMATION: TRANSDUCTION: all three about getting DNA into your organism. TRANSFECTION: Transformation is DNA into a prokaryote (except for yeast), Transduction is the use of infection (i.e. viruses) to get DNA inside, and Transfection is DNA into a eukaryote (except for yeast). With bugs: Two main techniques: CaCl2 and heat shock treatment and electroporation. In both cases need to make COMPETENT cells. Cells who are primed and ready for acceptance of DNA. Generally involves a series of growth steps so that bugs are at the just the right stage of growth. In CaCl method - competent cells have been treated with CaCl2. Essentially the membranes are thrashed around. BOTTOM LINE: cells are delicate. NOBODY REALLY KNOWS WHY or HOW CaCl2 + heat shock works, but it does. AND… it is easy to do Ice step is believed to allow the DNA to adhere to membrane. Heat shock may make the membrane move around more (fluid membrane). Membrane gets weaker, holes get bigger? DNA falls in? 37 incubation allows thrashed cells to recuperate. Also gives time for plasmid to replicate so that bugs are ampR Electroporation: will give much higher transformation efficiencies. ZAP a current through the bugs. Again nobody really knows whats going on. A bit more versatile (i.e. most organisms don't have a heat shock procedure). Microbiologists working on other bugs pseudomonads, bacilli, strept etc etc will use this piece of machinary. Competent cells are much easier to prepare. Procedure very quick. MORE EXPENSIVE… those cuvettes are generally not reusable, machine cost several thousand dollars. Different bugs use different setting, but the main idea is to vary amount of voltage applied. Dependant on the bug, the cuvette, etc etc. All of this will translate to some magic current value which is what causes this whole thing to work. BECAUSE OF THIS, you need to be careful with salt content in your cells or ligation mix. Making competent cells usually involves successive water washes. V=IR If you are not careful because of salt, or air pockets, etc etc.. You get something called ARCING. This is a small explosion happening, which usually consists of a bang (to varying degrees), cracking of the cuvette, a flash of light. General scariness but not at all dangerous. Transfection: getting DNA into eukaryotic cells: Most common method is still to use electroporation. ZAP the buggers Other alternatives include things like the Gene GUN (Ballistic approach), and viral mechanisms. 6.1 PROTEINS: General Info DAY 3 First off, some basic things to consider that ultimately affect how you envision the behaviour of protein in your experiments: 1. Proteins are not so simple Basic idea is that with DNA, you've got 4 different components (nucleotides), which are all essentially quite similar anyways. WITH proteins. 20 different amino acids. All with different sorts of properties, different sorts of charges, biochemical attributes. Consequently, proteins as a population are tough to predict. 2. Proteins can be high maintenance: Add to that the fact that proteins can often be high maintenance/delicate themselves, and you have an invitation for frustration. I.e. they degrade, enzymatically go off, they aggregate, they complex, etc. 3. Protein work still has lots of toys. (Although definitely not to the extent that DNA has. Also, one of the most powerful reagents you need – which may not be commercially available – is a specific antibody) 4. Protein work is more fun(?): interesting to note that when pressed for opinion, most will say that protein work tends to be more rewarding, interesting, and challenging. We’re going to focus on two techniques, the first of which (show Nature Table of Contents) will be the Western Blot analysis, and the second is the more fancy 2D gel electrophoresis analysis. BE CAREFUL keep things cold, but be sensitive to the structure of the proteins you work with – even cold temperatures can be detrimental to your experiments. QUANTITATION: (a few options) - Run a gel. Compare band with preweighed amounts of standard protein (like albumin) - works really well, kind of labour intensive. - USE colorimetric tests. BCA/Coomassie Blue tests works best. Easy to do, but can be very susceptible to chemicals that are commonly used in the buffers you store your proteins in (i.e. presence of detergents, tris, etc etc) - ABSORBANCE at 280nm. very rough 1.0 O.D. = 1mg/ml of protein (but as an example for IgG it’s closer to 1.4mg/ml)

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