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10 Things Every Molecular Biologist Should Know

Nick Oswald Suzanne Kennedy Megan Hogan Megan Cartwright

Edited By Nick Oswald

a BitesizeBio.com eBook Preface

Molecular is a funny old business...

Preface is a funny old One of the core aims of Bitesize Bio website yourself, or if you’d just like to let business. The ton of theoretical (www.bitesizebio.com) is to make it us know what you think (good or bad!) of knowledge that we cram in during our easier to learn on the job as a molecular our efforts, please feel free to get in touch undergrad years is scant preparation biologist by providing a place where via the contact page at BitesizeBio.com. for at the bench and leaves a lot to these nuggets of vital, often over-looked be said. knowledge can be found. Thanks for reading. When we first hit the lab there are so This short eBook provides 10 such many things to learn before we even nuggets, neatly contained in one place. get started that many things go From How SDS-PAGE works to the unlearned. chemical reason why enzymes have optimal temperatures we hope that there How specific techniques work, what The Bitesize Bio team. will be something in here that will actually is in those kits we use and why enlighten and entertain even the most we use certain approaches rather than experienced . others. If you like the information you find in this Often these nuggets of knowledge can eBook - which is the first of many we’ll mean the difference between be creating on a variety of topics - we understanding where an experiment is would love you pass the file onto your going wrong and not understanding, friends to help spread the word about between getting a result and not getting what we are doing at Bitesize Bio. one. And if you are hungry for more please be But normally, these nuggets can’t be sure to visit us at BitesizeBio.com to get found in lectures or in text books. the latest articles and eBooks we have Instead they must be gleaned through on offer. hard on-the-job experience or passed down by word-of-mouth from more Finally if you have any suggestions for experienced mentors. articles you’d like to see on Bitesize Bio, if you’d like to write something for the ii How SDS-PAGE Works

“If you are still wondering why the stacking gel is needed, think of what would happen if you didn’t use one.”

Section 01 By Nick Oswald SDS-PAGE (sodium dodecyl sulphate- charge is determined by amino acid charge of the protein. That’s where SDS polyacrylamide gel electrophoresis) is composition i.e. the sum of the positive comes in. commonly used in the lab for the and negative amino acids in the protein separation of proteins based on their and molecular radius by the protein’s The Role of SDS (et al) molecular weight. It’s one of those tertiary . techniques that is commonly used but not frequently fully understood. So let’s So in their native state, different proteins SDS is a detergent that is present in the try and fix that. with the same molecular weight would SDS-PAGE sample buffer where, along migrate at different speeds in an with a bit of boiling, and a reducing agent SDS-PAGE separates proteins electrical field depending on their charge (normally DTT or B-ME to break down according to their molecular weight, and 3D shape. protein-protein disulphide bonds), it based on their differential rates of disrupts the tertiary structure of proteins. To separate proteins in an electrical field migration through a sieving matrix (a This brings the folded proteins down to based on their molecular weight, we gel) under the influence of an applied linear molecules. electrical field. need to destroy the tertiary structure by reducing the protein to a linear molecule, Making the rate of protein and somehow mask the intrinsic net migration proportional to molecular weight.

The movement of any charged species through an electric field is determined by it’s net charge, it’s molecular radius and the magnitude of the applied field. But the problem with natively folded proteins is that neither their net charge SDS helps to disrupt the tertiary nor their molecular radius are molecular protein structure weight dependent. Instead, their net

iv SDS also coats the protein with a The gel matrix be made up at a variety of concentrations uniform negative charge, which masks to produce different pore sizes which, in the intrinsic charges on the R-groups. turn, can produce varying separating SDS binds fairly uniformly to the linear In an applied electrical field, the SDS- conditions that can be adjusted to suit your proteins (around 1.4g SDS/ 1g protein), treated proteins will now move toward needs. meaning that the charge of the protein the cathode at different rates depending is now approximately proportional to it’s on their molecular weight. These The discontinuous buffer molecular weight. different mobilities will be exaggerated system and the stacking gel by the high-friction environment of a gel SDS is also present in the gel to make matrix. sure that once the proteins are To conduct the current through from the linearized and their charges masked, As the name suggests, the gel matrix anode to the cathode through the gel, a they stay that way throughout the run. used for SDS-PAGE is polyacrylamide, buffer is obviously needed. Mostly we use which is a good choice because it is the discontinuous Laemmli buffer system. The dominant factor in determining the chemically inert and, crucially, can easily migration rate of an SDS-coated protein “Discontinuous” simply means that the in a gel is it’s molecular radius. buffer in the gel and the tank are different. SDS-coated proteins have been shown Typically, the system is set up with a to be linear molecules, 18 Angstroms stacking gel at pH 6.8, buffered by Tris- wide and with length proportional to HCl, a running gel buffered to pH 8.8 by their molecular weight, so the molecular Tris-HCl and an electrode buffer at pH 8.3. radius (and hence their mobility in the The stacking gel has a low concentration of gel) is determined by the molecular acrylamide and the running gel has a weight of the protein. Since the SDS- higher concentration capable of retarding coated proteins have the same charge the movement of the proteins. to mass ratio there will be no differential migration based on charge. So what’s with all of those different pH’s? Well, glycine can exist in three different The discontinuous buffer system charge states, positive, neutral or negative

v depending on the pH. This is shown in All of the proteins in the gel sample have So the glycine front accelerates past the the diagram below. Control of the an electrophoretic mobility that is proteins, leaving them in the dust. charge state of glycine by the different intermediate between the extreme of the The result is that the proteins are dumped buffers is the key to the whole stacking mobility of the glycine and Cl- so when in a very narrow band at the interface of the gel thing. the two fronts sweep through the stacking and running gels and since the sample-well the proteins are So here’s how the stacking gel works. running gel has an increased acrylamide concentrated into the narrow zone When the power is turned on, the concentration, which slows the movement between the Cl- and glycine fronts. negatively-charged glycine ions in the of the proteins according to their size, the pH 8.3 electrode buffer are forced to And they’re off! separation begins. enter the stacking gel, where the pH is 6.8. In this environment glycine What was all of that about? switches predominantly to the This procession carries on until it hits the zwitterionic (neutrally charged) state. running gel, where the pH switches to If you are still wondering why the stacking This loss of charge causes them to 8.8. At this pH the glycine molecules are gel is needed, think of what would happen move very slowly in the electric field. mostly negatively charged and can if you didn’t use one. migrate much faster than the proteins. The Cl- ions (from Tris-HCl) on the other hand, move much more quickly in Gel wells are around 1cm deep and you the electric field and they form an ion generally need to substantially fill them to front that migrates ahead of the glycine. get enough protein onto the gel. So in the The separation of Cl- from the Tris absence of a stacking gel, your sample counter-ion (which is now moving would sit on top of the running gel, as a towards the cathode) creates a narrow band of up to 1cm deep. zone with a steep voltage gradient that Rather than being lined up together and pulls the glycine along behind it, hitting the running gel together, this would resulting in two narrowly separated mean that the proteins in your sample The effect of buffer pH on glycine fronts of migrating ions; the highly would all enter the running gel at different charge mobile Cl- front, followed by the slower, times, resulting in very smeared bands. mostly neutral glycine front.

vi So the stacking gel ensures that all of the proteins arrive at the running gel at the same time so proteins of the same molecular weight will migrate as tight bands. Separation

Once the proteins are in the running Did you find this useful? gel, they are separated because higher molecular weight proteins move more Visit BitesizeBio.com for many more slowly through the porous acrylamide eBooks, articles, webinars and videos on gel than lower molecular weight topics just like this one. proteins. The size of the pores in the gel can be altered depending on the size of the proteins you want to separate by changing the acrylamide concentration.

For a broader separation range, or for proteins that are hard to separate, a gradient gel, which has layers of increasing acrylamide concentration, can be used.

vii How Proteases and Protease Inhibitors Work

“To defeat the protease, you must become the protease”

Section 02 By Megan Cartwright Proteases: wild, mysterious, active site and how it interacts with the To go back to the serine example, our destructive. What are these untamed target peptide bond. friend trypsin hydrolyzes on the carboxyl elements ravaging your precious side of positively charged arginine or lysate? Camp One hydrolyzes the bond through lysine, unless these are followed by a nucleophilic activation of a serine, proline. How can a drop of EDTA or a smidge of threonine, or cysteine at its active site. “cocktail” protect that sample, which is And while its buddy chymotrypsin is also a gently cradling your hopes, your Camp Two, which includes the aspartic, serine protease, chymotrypsin simply dreams, and your desire to survive the glutamic, and metalloproteases, uses doesn’t touch arginine or lysine and really next lab meeting? water itself for the hydrolysis. just prefers aromatics like tyrosine, tryptophan, or phenylalanine. Brace yourself for a biochem flashback: These tidy camps break down under in this section, I’ll explain the what and questioning about preferred targets and The last thing a wants is these how of proteases and protease substrate affinity. Although Camp One’s enzymes running amok, rampaging willy- inhibitors, and we’ll do it with phrases serine proteases all agree that an active nilly through the cytoplasm, chewing off- like “reversible competitive inhibition” site serine is superior to the plebeian target, and generally behaving like – I say and “zymogen.” cysteine, they still have tense family this with great familiarity – Ohio State dinners because of each of the football fans. Proteases: the good, the proteases has a different target preference and structure. Regulation and segregation are the cell’s bad, the hydrolytic prime approaches for dealing with these These structural differences drive a enzymes: regulation by, for example, protease’s specificity for where it will All have proteolytic and holding them in an inactive precursor state attack: terminal hydrolysis for the phosphorylating enzymes. You. Me. (a “zymogen”) and segregation to the exopeptidases, internal hydrolysis for the Helicoverpa armigera. While involved lysosome or secretory pathways, among endopeptidases. in everything from host defense to other locations. wound healing to viral replication, Although the exopeptidases only choose proteases can be broadly lumped into between the amino or carboxyl terminus, two camps based on what’s in their the endopeptidases are gloriously picky.

ix Inhibitors: You can’t stop and can be anything from small enhanced affinity for metal ions to complex molecules like sodium fluoride to big with them so that nobody else can react with just one honkin’ polypeptide analogues of with them. This matters because ’s own microbial inhibitors. metalloproteases need zinc, magnesium, All that chemical lysis, scraping, They can compete with substrates for an manganese, calcium, or even cobalt in their sonication, and freeze-thawing you active site – sometimes destroying the active site to activate water to hydrolyze inflict throws the whole system into site in the process – or selfishly hoard peptide bonds to meet their quarterly chaos. No peptide bond is safe! the precious, precious cations needed hydrolysis goals. Take away that zinc with for protease function. EDTA, complex up that calcium with EGTA, Exopeptidases attack the ends of and it’s bye-bye metalloprotease activity. amino acid chains while Competitive inhibitors act by binding in a endopeptidases hydrolyze their internal substrate-like manner to the active site to Conclusion: To defeat the block it or by binding and destroying it links. Kinases phosphorylate and protease, you must become dephosphorylate at will. Good luck through modifications such as quantifying NOS induction or making a esterification. Many large competitive the protease case for p53 activation by Western. inhibitors boost their affinity and Without inhibitors, your results will be specificity by binding to the overlooked inconsistent, your lab meetings wallflower regions neighboring the hogs- Proteases are just some of the destructive agonizing, and your graduation date a all-the-glory active site. You have likely forces aims at your beloved cell pipe dream. already come across competitive lysates, and protease inhibitors are one of inhibitors. They include all sorts, from the ways that the clever scientist can, judo- Tragically, there is no Wunderkind aprotinin, a 6 kDa polypeptide serine like, turn the universe against itself. For inhibitor that takes out all proteases, protease that is reversible if you ask more information on beating the universe halts every phosphorylation event, and politely at pH extremes, to sodium into submission, check out Emily Crow’s does some light tidying-up about the orthovanadate, a small molecule which “How To Preserve Your Samples In centrifuges. It takes a “cocktail” of inhibits the ever-gullible protein Western Blotting”, Jode Plank’s “Streamline approaches to stave off proteolysis. phosphotyrosyl phosphatases. Your Western Blots”, on Bitesize Bio. Protease inhibitors can act irreversibly, reversibly, and reversibly-under-duress, By contrast, the toddler-like chelators (motto: MINE!) take advantage of their

x Agarose Gels Do Not Polymerize

“Polyacrylamide gel polymerization - Try saying that three times quickly!”

Section 03 By Nick Oswald I was browsing a certain website the chemically inert – so won’t interact with them and causing them to react with each other day when I came across a proteins as they pass through – and that other to form the polyacrylamide chain. protocol that advised: “When agarose it can be easily and reproducibly made In the absence of bis-acrylamide, the starts to cool, it undergoes what is with different pore sizes to produce gels acrylamide would polymerize into long known as polymerization” with different separation properties. strands, not a porous gel. But as the Polymerizing agarose? Someone is The polymerization reaction, shown in diagram shows, bis-acrylamide cross-links getting their gel matrices mixed up, and the diagram below, is a vinyl addition the acrylamide chains and this is what it’s not the first time I have heard this catalyzed by free radicals. The reaction gives rise to the formation of the porous gel said, so it looks like some myth-busting is initiated by TEMED, which induces matrix. The amount of cross-linking, and is required. free radical formation from ammonium therefore the pore size and consequent persulphate (APS). The free radicals separation properties of the gel can be So hunker down for a quick run down transfer electrons to the acrylamide/ controlled by varying the ratio of acrylamide on the difference between polymerizing bisacrylamide monomers, radicalizing to bis-acrylamide. and non-polymerizing gel matrices.

Of the common gel matrices used in molecular biology, polyacrylamide, agar and agarose, polyacrylamide is the one that polymerizes. I’ll deal with agarose and agar later. Polyacrylamide gel polymerization

Polyacrylamide, used mainly for SDS- PAGE, is a matrix formed from monomers of acrylamide and bis- Polyacrylamide gel polymerization - Try saying that three times quickly! acrylamide. It’s strengths are that is it

xii For more information on polyacrylamide So agarose (and agar) gels form by gel polymerization see Biorad Bulletin gellation through hydrogen bonding 1156 and electrostatic interactions, not through polymerization. Agarose gel formation

So what about agarose? Well, agarose – the main component of the gelatinous agar that can be isolated from certain species of seaweed – is itself a polymer. But, polymerization is not the Did you find this useful? mechanism for agarose gel formation.

Chemically, agarose is a Visit BitesizeBio.com for many more polysaccharide, whose monomeric unit eBooks, articles, webinars and videos on is a disaccharide of D-galactose and topics just like this one. 3,6-anhydro-L-galactopyranose which is shown in the diagram below. Chemically, agarose is a In aqueous solutions below 35°C these polysaccharide, whose polymer strands are held together in a monomeric unit is a disaccharide porous gel structure by non-covalent of D-galactose and 3,6-anhydro-L- interactions like hydrogen bonds and galactopyranose electrostatic interactions. Heating the solution breaks these non-covalent interactions and separates the strands. Then as the solution cools, these non- covalent interactions are re-established and the gel forms.

xiii How Ethanol Precipitation of DNA and RNA Works

“First we need to know why nucleic acids are soluble in water...”

Section 04 By Nick Oswald Ethanol precipitation is a commonly hydrogen atoms (see the diagram molecules, which can’t easily interact with used technique for concentrating and below). water molecules, are hydrophobic. de-salting (DNA or RNA) Nucleic acids are hydrophilic due to the preparations in aqueous solution. The Because of these charges, polar negatively charged phosphate (PO3-) basic procedure is that salt and ethanol molecules, like DNA or RNA, can interact groups along the sugar phosphate are added to the aqueous solution, electrostatically with the water backbone. which forces the nucleic acid to molecules, allowing them to easily dissolve in water. precipitate out of solution. The role of the salt… The precipitated nucleic acid can then Polar molecules can therefore be be separated from the rest of the described as hydrophilic and non-polar solution by centrifugation. The pellet is Ok, so back to the protocol. The role of the washed in cold 70% ethanol then after salt in the protocol is to neutralize the a further centrifugation step the ethanol charges on the sugar phosphate backbone. is removed, and the nucleic acid pellet A commonly used salt is sodium acetate. In is allowed to dry before being solution, sodium acetate breaks up into Na resuspended in clean aqueous buffer. + and [CH3COO]-.

So how does this work? The positively charged sodium ions neutralize the negative charge on the PO3- A bit about solubility… groups on the nucleic acids, making the molecule far less hydrophilic, and therefore much less soluble in water. First we need to know why nucleic acids are soluble in water. Water is a The role of the ethanol… polar molecule – it has a partial Polarity of water molecules. Oxygen negative charge near the oxygen atom atoms are slightly negatively charged, The electrostatic attraction between the due the unshared pairs of electrons, hydrogens are slightly positive and partial positive charges near the Na+ ions in solution and the PO3- ions are dictated by Coulomb’s Law, which is

xv affected by the dielectric constant of the The wash step with 70% vitro translation or reverse transcription. In solution. these cases, use NaAc. ethanol… Water has a high dielectric constant, ▪ Use Ammonium acetate (2M final conc) which makes it fairly difficult for the Na+ for the removal of dNTPs, but do not use and PO3- to come together. This step is to wash any residual salt for preparation of DNA for T4 away from the pelleted DNA. polynucleotide kinase reactions as Ethanol on the other hand has a much ammonium ions inhibit the enzyme. lower dielectric constant, making it A few tips on nucleic acid much easier for Na+ to interact with the precipitation… To increase the yield in precipitations of PO3-, shield it’s charge and make the low concentration or small nucleic acid nucleic acid less hydrophilic, causing it pieces (< 100 nucleotides) to drop out of solution. Choice of salt ▪ Add MgCl2 to a final concentration of The role of temperature… ▪ Use Sodium acetate (0.3M final conc, 0.01M pH 5.2) for routine DNA precipitations ▪ Increase the time of incubation on ice before centrifugation to 1 hour. Incubation of the nucleic acid/salt/ ▪ Use Sodium chloride (0.2M final conc) ethanol mixture at low temperatures for DNA samples containing SDS since (e.g. -20 or -80C) is commonly cited in NaCl keeps SDS soluble in 70% ethanol protocols as necessary in protocols. so it won’t precipitate with the DNA. Did you find this useful?

However, according to Maniatis et al ▪ Use Lithium Chloride (0.8M final conc) for RNA. This is because 2.5-3 volumes (Molecular Cloning, A Laboratory Visit BitesizeBio.com for many more of ethanol should be used for RNA Manual 2nd Edition… 2nd edition?? – I eBooks, articles, webinars and videos on precipitation and LiCl is more soluble in need to get a newer version!), this is topics just like this one. not required, as nucleic acids at ethanol than NaAc so will not precipitate, concentrations as low as 20ng/mL will but beware – chloride ions will inhibit precipitate at 0-4C so incubation for protein synthesis and DNA polymerase 15-30 minutes on ice is sufficient. so LiCl is no good for RNA preps for in

xvi How Alkaline Lysis Works

“The easiest way to describe how alkaline lysis works is to go through the procedure and explain each step, so here goes....”

Section 05 By Nick Oswald Alkaline lysis was first described by bacterial cell wall. EDTA chelates denatures most of the proteins in the cells, Birnboim and Doly in 1979 (Nucleic divalent cations in the solution which helps with the separation of the Acids Res. 7, 1513-1523) and has, with preventing DNases from damaging the proteins from the plasmid later in the a few modifications, been the preferred plasmid and also helps by destabilizing process. method for plasmid DNA extraction the cell wall. Glucose maintains the It is important during this step to make sure from ever since. The easiest osmotic pressure so the cells don’t burst that the re-suspension and lysis buffers are way to describe how alkaline lysis and RNase A is included to degrade well mixed, although not too vigorously. works is to go through the procedure cellular RNA when the cells are lysed. and explain each step, so here goes. Also remember that SDS and NaOH are 3. Lysis pretty nasty so it’s advisable to wear gloves 1. and and eye protection when performing Harvesting The lysis buffer (aka solution 2) contains alkaline lysis. sodium hydroxide (NaOH) and the detergent Sodium Dodecyl (lauryl) 4. Neutralization The procedure starts with the growth of Sulfate (SDS). the bacterial cell culture harboring your Addition of potassium acetate (solution 3) plasmid. When sufficient growth has SDS is there to solubilize the cell increases the pH of the mixture. Under been achieved, the cells are pelleted by membrane. NaOH helps to break down these conditions the hydrogen bonding centrifugation to remove them from the the cell wall, but more importantly it between the bases of the single stranded growth medium. disrupts the hydrogen bonding between DNA can be re-established, so the ssDNA the DNA bases, converting the double- can re-nature to dsDNA. 2. Re-suspension stranded DNA (dsDNA) in the cell, including the genomic DNA (gDNA) and This is the selective part. While it is easy The pellet is then re-suspended in a your plasmid, to single stranded DNA for the the small circular plasmid DNA to re- solution (normally called solution 1, or (ssDNA). nature it is impossible to properly anneal similar in the kits) containing Tris, those huge gDNA stretches. This process is called denaturation and EDTA, glucose and RNase A. Divalent is central part of the procedure (which is This is why it’s important to be gentle cations (Mg2+, Ca2+) are essential for why it’s called alkaline lysis!). SDS also during the lysis step because vigorous DNase activity and the integrity of the xviii mixing or vortexing will shear the gDNA followed by ethanol precipitation and producing shorter stretches that can re- affinity chromotography-based methods anneal and contaminate your plasmid using a support that preferentially binds prep. to the plasmid DNA under certain conditions of salt or pH, but releases it While the double-stranded plasmid can under other conditions. The most dissolve easily in solution, the single common methods are detailed in the stranded genomic DNA, the SDS and Bitesize Bio article on 5 ways to clean up the denatured cellular proteins stick a DNA sample. together through hydrophobic interactions to form a white precipitate. The precipitate can easily be separated from the plasmid DNA solution by centrifugation. Did you find this useful? 5. Cleaning and concentration Visit BitesizeBio.com for many more eBooks, articles, webinars and videos on topics just like this one. Now your plasmid DNA has been separated from the majority of the cell debris but is in a solution containing lots of salt, EDTA, RNase and residual cellular proteins and debris, so it’s not much use for downstream applications. The next step is to clean up the solution and concentrate the plasmid DNA.

There are several ways to do this including phenol/chloroform extraction

xix How Silica Spin Column DNA and RNA Preps Work

“...the downside [of using kits] is that we don't actually know what is in the mysterious... set of solutions which... makes troubleshooting more difficult.”

Section 06 By Suzanne Kennedy These days, most labs use commercial Lysis the protein, the better Proteinase K works. kits, which employ spin columns, for the Lysozyme, however, does not work in the isolation of nucleic acids. The spin denaturing conditions of the lysis buffer, so columns contain a silica resin that lysozyme treatment is usually done before The lysis approach may vary based on selectively binds DNA/RNA, depending adding the denaturing salts. the whether you want DNA or RNA, but on the salt conditions and other factors the common denominator is a lysis buffer For plasmid preps, the lysis is very different influenced by the extraction method. containing a high concentration of from extraction for RNA or genomic DNA When things are going well, these kits chaotropic salt. because the plasmid has to be separated make the whole process much easier from the genomic DNA first. Chaotropes destabilize hydrogen bonds, and faster than the 'Methods of Old'. van der Waals forces and hydrophobic If you throw in chaotropes, you’ll release However, the downside is that we don't interactions. Proteins are destabilized, everything at once and won’t be able to actually know what is in the mysterious including nucleases, and the association differentially separate the small circular and proprietary set of solutions which, of nucleic acids with water is disrupted DNA from the high molecular of course, makes troubleshooting more setting up the conditions for their transfer weight chromosome. difficult. to silica. So in plasmid preps the chaotropes are not In this section I’ll explain how silica spin Chaotropic salts include guanidine HCL, added until after lysis and the salts are filter kits work and what is going on at guanidine thiocyanate, urea, and lithium used for binding. each step. perchlorate. A more in-depth explanation of alkaline I’ll also go over some common Besides the chaotropes, lysis buffer lysis for plasmid preps can be found in the problems specific to silica columns that usually contains detergents to help with previous section “How Alkaline Lysis can be overcome or avoided with just a protein solubilization and lysis. Works” little extra understanding. There can also be enzymes used for lysis depending on the sample type. Proteinase K is one of these, and actually works very well in these denaturing buffers; the more denatured

xxi Binding and want to troubleshoot recovery, this The wash steps serve to remove these can be a step to evaluate further. impurities. There are typically two washes, although this can vary depending on the Another way to diagnose problems is to The chaotropic salts are critical for sample type. The first wash will often have save the flow-through after binding and lysis, but also for binding of the nucleic a low amount of chaotropic salt to remove precipitate it to see if you can find the acids to the silica resin. the protein and colored contaminants. This nucleic acids you are searching for. If is always followed with an ethanol wash to To enhance the binding of nucleic acids you used an SDS-containing detergent in remove the salts. If the prep is something to silica, alcohol is also added at the lysis, try using NaCl as a precipitant to that didn’t have a lot of protein to begin binding stage. Most of the time this is avoid contamination of the DNA or RNA with, such as plasmid preps or PCR clean ethanol but sometimes it may be with detergent. up, then only an ethanol wash is needed. isopropanol. Washing Steps Removal of the chaotropic salts is crucial to The concentration of ethanol, and getting high yields and purity of DNA or the volume used during binding, both RNA. Some kits will even recommend have big effects. Too much Your lysate was centrifuged through the washing the column twice with ethanol. If (concentration or volume) and you’ll silica membrane and now your DNA or salt remains behind, the elution of nucleic bring in a lot of degraded nucleic acids RNA should be bound to the column and acid is going to be poor, and the A230 and small species that will influence the impurities, protein and reading will be high, resulting in low UV260 readings and throw off some polysaccharides, should have passed 260/230 ratios. of your yields. Too little, and it may through. become difficult to wash away all of the Dry Spin salt from the membrane. But, the membrane is still dirty with residual proteins and salt. If the The important point here is that the sample was from , there will still be ethanol influences binding and the polysaccharides, maybe some pigments After the ethanol wash, most protocols amount added is optimized for too, left on the membrane, or if the have a centrifugation step to dry the whatever kit you are using. Modifying sample was blood, the membrane might column. This is to remove the ethanol and that step can help change what you be tinted brown or yellow. is essential for a clean eluant. When 10 recover so if you are having problems mM Tris buffer or water is applied to the

xxii membrane for elution, the nucleic acids rehydrate in the short time used for Make sure to use fresh high quality ethanol can become hydrated and will release elution. Elution of DNA can be (100% 200 proof) to dilute buffers or for from the membrane. If the column still maximized by allowing the buffer to sit in adding to the binding step. has ethanol on it, then the nucleic acids the membrane for a few minutes before Low quality ethanol or old stocks may have cannot be fully rehydrated. centrifugation. taken on water and not be the correct Skipping the drying step results in RNA, on the other hand, is fine at a concentration. If the wash buffer is not ethanol contamination and low yields. slightly acidic pH and so water is the made correctly, you may be washing off preferred diluent. RNA dissolves readily your DNA or RNA. The main indication of this problem in water. comes when you try to load the sample Low Purity: If the sample is contaminated onto an agarose gel; the DNA will not with protein (low 260/280) then maybe you sink, but floats in the buffer, even in the started with too much sample and the presence of loading dye. Another What other things can go protein was not completely removed or indicator of ethanol contamination is dissolved. that if you put the sample in the -20C, it wrong with silica spin If the sample has poor 260/230 ratio the doesn’t freeze. columns? issue is usually salt from the bind or the Elution wash buffer. Make sure that the highest quality ethanol was used to prepare wash Low yields: If you experience yields buffers and if the problem continues, give The final step is the release of pure lower than you expected for a sample, the column an additional wash. DNA or RNA from the silica. For DNA there are many factors to think about. Some samples have a lot more inhibitors preps, 10 mM Tris at a pH between 8-9 Usually it is a lysis problem, and compared to others. Environmental is typically used. DNA is more stable at incomplete lysis is a very common cause samples are especially prone to purity a slightly basic pH and will dissolve of low yields. issues because humic substances are faster in a buffer. This is true even for solubilized during extraction. Humics DNA pellets. Water tends to have a low Alternatively, the problem could be behave similarly to DNA and are difficult to pH, as low as 4-5 and high molecular incorrect binding conditions. remove from the silica column. For this weight DNA may not completely type of sample, specialized techniques xxiii exist to remove the protein and humics question I ask people is “did you check prior to the column step. the results of the PCR on a gel?” because you cannot UV check a PCR Degradation: This is more of a reaction and get an accurate reading. concern for RNA preps and an article that gives specific advice can be found There is way too much in a PCR reaction in the Bitesize Bio article absorbing UV at 260: nucleotides, “Troubleshooting RNA Isolation”. detergents, salts, and primers. In my If you liked this, you’ll experience, apparent failure of a PCR Mainly with RNA, degradation occurs also like: clean-up kit to work frequently is from improper storage of the sample or normally caused by a PCR reaction that an inefficient lysis, assuming of course has failed and so there was nothing to that you eluted with RNase-free water. Recycle Those DNA Extraction clean up in the first place. For DNA preps, degradation is not a Columns huge problem because for PCR, the But if you know you had a strong PCR DNA can be sheared and it works fine. product, the best approach is to just save by Liam Thompson at BitesizeBio.com But if you were hoping not to have so your flow-through fraction after binding. If much sheared DNA, then you may the DNA doesn’t bind, that’s where it is. have used too strong a lysis method. You can always rescue it and then clean You know those ridiculously priced and it up again. And then call tech support throw-away DNA mini, midi and maxi-prep PCR Clean-up Special and ask for a replacement kit. columns? Well the good news is that you Considerations: PCR clean-up is the can actually re-use them if you are easiest of all silica spin column reasonably careful at regenerating them, techniques, because it simply involves with this simple and cheap method.. adding a high concentration of binding salts (typically between 3-5 volumes of ...[click to read more] salt per volumes of PCR reaction) and centrifuging the column.

So when PCR Clean-up kits fail, it can be particularly frustrating. The first xxiv How DNA Ligation Works

“Don’t be scared off by the , this is worth taking some time to understand!”

Section 07 By Nick Oswald DNA ligase (EC 6.5.1.1) covalently Why? Well, as you will probably know, all The second step is the enzymatic reaction, joins the phosphate backbone of DNA molecules move faster at higher which is shown schematically on the next with blunt or compatible cohesive ends temperatures so you can imagine that it page. (see figure below) and it’s natural role is going to be easier for two DNA ends to DNA ligase catalyzes the joining of the 3′- is in repairing double strand breaks in collide and stay together if they are OH to the 5′-phosphate via a two step DNA molecules. gently floating through the solution at low mechanism. First, the AMP nucleotide, temperature, rather than whizzing about In molecular biology it is commonly which is attached to a lysine residue in the as they would be at higher temperatures. used for the insertion of restriction enzyme’s active site, is transferred to the enzyme-generated DNA fragments into For cohesive ends, there is an additional 5′-phosphate. Then the AMP-phosphate vector backbones. Commercial ligases reason; lower temperatures stabilize the bond is attacked by the 3′-OH, forming the are supplied with a reaction buffer hydrogen bonding between the covalent bond and releasing AMP. containing ATP and Mg2+, which are complementary nucleotides, which really To allow the enzyme to carry out further both essential for ligase activity. helps to keep things in place. reactions the AMP in the enzyme’s active Since ATP can be damaged by repeated freeze-thaw cycles, it is advisable to make aliquots of the buffer (see the Bitesize Bio article “5 DNA ligation tips” for more information).

The ligation reaction itself has two basic steps. Firstly the DNA ends have to collide by chance and stay together long enough for the ligase to join them.

This is the most inefficient part of the reaction, but is easier at low The DNA ligase can covalently join blunt ends, but cohesive ends are a bit easier temperatures. because the hydrogen bonds hold the cohesive ends in the right place for the enzyme to do the job.

xxvi site must be replenished by ATP.

The DNA ligase enzyme has optimal activity at 25°C so the ligation reaction is carried out at a temperature that is a trade-off between the optimal temperatures for bringing the DNA ends together (1°C) and the enzymatic reaction (25°C). Normally 1hr at 16°C is fine but since bringing the DNA ends together is the least efficient part of the reaction favoring this by lowering the temperature to 4°C can give even more efficiency.

However, the enzyme will work very slowly at this temperature so a long The DNA ligation mechanism. Don’t be scared off by the chemistry, this is worth (e.g. overnight) incubation time is taking some time to understand! required.

If you liked this, you’ll also like:

Pinpointing DNA Ligation Problems

by Nick Oswald at BitesizeBio.com

xxvii How Colorimetric Assays Work

“...even the most traditional of molecular biologists perform chemical reactions nearly every day”

Section 08 By Megan Hogan One of my colleagues, a very good performing these assays and Kinases and phosphatases mediate a molecular biologist, told me that the understanding the results, as well as in variety of cellular processes like only time she uses chemistry is when troubleshooting when things go awry. , gene transcription and she needs to calculate molarities. translation, protein-protein interactions, and By taking advantage of the electronic apoptosis, through protein phosphorylation I, of course, scoffed at this statement, properties and specific wavelengths of and dephosphorylation. For this reason, and tried to remind her of all the chromophores, we can deduce a great many different technologies, including chemistry she uses daily. deal of information. Colorimetric assays various colorimetric assays, exist to detect help us to distinguish a particular I may be a bit biased since I am a these enzymes and their activity. enzyme from another, to quantitate chemist, but surely, all the catalytic activity, as well as inhibition of chromophores, fluorophores, and para-Nitrophenylphosphate this activity, to generate proliferative and imaging probes, etc., involve some Assay Mechanism profiles, and even to determine knowledge of chemistry and chemical the concentration of proteins in solution. para-Nitrophenylphosphate (pNPP) is reactions. She shrugged, and replied They are a simple and convenient way to widely used as a synthetic substrate to that they’re all supplied in kits. visualize biological processes. As a measure the catalytic activity of various So, in defense of all chemistry nerds former PI of mine used to say, in his thick phosphatases. out there, here is my rebuttal: even the German accent, while pointing to his The phosphate group of pNPP is cleaved most traditional of molecular biologists eyes, “Nature has provided us with our by the enzyme to yield p-nitrophenol, which perform chemical reactions nearly own spectrophotometers, no?” is also colorless since the wavelength every day. Even more appealing, many colorimetric maximum for the electronic excitation of p- From purification by ion exchange assays are commercially available as nitrophenol in water is 318 nm. However, resins and affinity columns, to kits, usually with a detailed protocol. In under alkaline conditions, the p-nitrophenol calculating protein concentrations and fact, colorimetric assays are used so is converted to the p-nitrophenolate anion, coupling bioconjugates, chemistry is regularly in chemistry and biology, that to resulting in a bathochromic shift (a change behind it all. For this article I will focus cover them all would take an entire in absorbance of a compound to a longer on a couple of colorimetric assays and book. So, for simplicity’s sake, this wavelength based on solution conditions) hopefully convince you that knowledge article will focus on two phosphatase to around 400 nm. This is at the blue edge of these chemical reactions is helpful in assays. of the electromagnetic spectrum, but since xxix we see the color of the reflected light In that instance, the end point addition of formed between malachite green, (as opposed to the absorbed light), the a strong base (such as sodium hydroxide ammonium molybdate, and free solution looks yellow. or potassium hydroxide) is necessary in orthophosphate (aka inorganic phosphate, order to obtain the desired yellow color. Pi) under acidic conditions. Things to Keep in Mind… Orthophosphate, liberated from a One of the great selling points of the Malachite Green phosphorlyated substrate upon cleavage pNPP assay is its simplicity, so it’s easy Assay Mechanism by the phosphatase, forms a complex with to see why one might forget that this The para-nitrophenylphosphate assay is ammonium molybdate in a solution of bathochromic (a great trivia word!) shift great, but what if your favorite sulfuric acid. The formation of the is a crucial element to the assay. phosphatase doesn’t efficiently malachite green phosphomolybdate What if, for example, the enzymatic metabolize pNPP? Another commercially complex, measured at 620-650 nm, is activity requires an optimum pH in the available phosphatase assay is the accordingly directly related to the free acidic range, as so happens to be the malachite green assay. This simple orthophosphate concentration. Therefore, it case with acid phosphatase? assay method is based on the complex is possible to quantify phosphorylation and phosphate release from protein phosphatase substrates.

Things to Keep in Mind…

This assay measures only inorganic free phosphate; organophosphates (lipid-bound or protein-bound phosphates) must first be hydrolyzed and neutralized prior to measurement. Knowledge of the different classes of organophosphates and their respective different free energies of hydrolysis aids in the optimization of assay para-Nitrophenylphosphate: From Colorless to Yellow conditions. Higher-energy organophosphates (acetal phosphates,

xxx phosphoanhydrides, etc) possess acid- ion associate in the aqueous solution, There’s much more where labile phosphate groups that can be polyvinylalcohol is added to the solution. released into solution by incubation at those came from low pH. On the other hand, lower- Another thing to take into consideration during the assay is the possibility of any energy organophosphates These two examples barely skim the redox reactions that could interfere with (phosphoesters) are stable in acidic surface of the colorimetric assays. Just the assay. Molybdenum is a transition solution and require much harsher think of all the catalase assays for the metal and so exists in many oxidation conditions (such as thermal detection of hydrogen peroxide (like states. In the molybdate anion, it has an decomposition) to be detected by this Sigma’s quioneimine derived assay), and oxidation state of +6. Reduction of the method. of course, the very popular MTT-based acidified Mo(VI) solution by organic assays for measuring cytotoxicity. A better In the presence of a large excess of compounds, like ascorbic acid and understanding of the chemistry involved in malachite green, the 3:1 ion associate reducing sugars (i.e. glucose), or by these assays helps to provide a deeper ((MG+)3(PMo12O40 3-)) can easily inorganic compounds, like SnCl2, understanding of the reactions, as well as form and precipitate in the acidic creates the Mo(IV) species, which is blue any required troubleshooting procedures. aqueous solution. To stabilize the 1:1 in color. Colorimetric assays are just one of the many ways in which chemistry and biology work hand-in-hand in scientific research. I think it’s fair to say that the multi- disciplinary approach is now recognized as a vital way to move forward in the drug discovery process.

My professor commented that in the ‘olden days’ it was enough to study medicinal chemistry and that the “No sleep ‘til 640nm”, By Malachite Green pharmaceutical companies would teach you the rest. Now, medicinal chemists are

xxxi expected to understand , Want More Chemistry for Biologists? , and molecular biology techniques. It takes an entire team of Take a look at this free webinar series from Peter Kirsop at BitesizeBio.com people, involving many specialities, in order to be successful in developing safe and effective drugs. But it is not Chemistry for Biologists enough simply to involve many collaborators; I would argue that Covered in this series: researchers on all sides need to learn Biological Organic Molecules to occasionally think alike. So hopefully The Importance of Stereochemistry in Biological Molecules the next time my aforementioned colleague performs a colorimetric assay Features of Organic Molecules she can don her ‘chemistry hat’ in addition to her ‘molecular biology hat’, and remember that drug research is an interdisciplinary art.

xxxii How Lab Grade Water is Purified

“...in practice a number of techniques are used, each of which can remove a different set of impurities.”

Section 09 By Nick Oswald There’s something in the water, and it with a boiling point equal to or less than 5. Filtration through a bed of activated would love to go after your that of water will also be carried over in carbon is useful for removing things like experiments. to the distillate. chloride ions and organic compounds, which are adsorbed onto the surface of the Straight out of the tap, water contains 2. Microfiltration. In this technique, carbon. , endotoxins, DNase pressure is used to force the water and RNase, salts and other impurities through a filter with pore sizes of 1 to 0.1 6. UV radiation. We all know what UV that could gobble up your experiment in micron in order to remove particulate radiation, at specific wavelengths, can do one bite. matter. Filter diameters lower than 0.2 to DNA and microorganisms. So UV is an micron removes bacteria – so-called cold obvious way to remove microorganisms Of course we avoid this drama sterilization. from the water. It can also clean up the completely by using purified water from water by breaking down certain organic which these nasties have been 3. Ultrafiltration uses even smaller pore compounds into less harmful products. removed. But how is this purification sizes (down to 0.003 micron). These are done? essentially molecular sieves, which 7. Deionization/ Ion exchange. This remove molecules with a diameter larger technique removes ions from the water by Well in practice a number of techniques than the pore size. It can be used to passing it through a resin bed containing a are used, each of which can remove a remove , endotoxins, RNase and mixture of cationic and anionic resins. different set of impurities. DNase Positive ions in the water are attracted to So here are the the anionic resin particles and negative 4. Reverse osmosis. If you thought that ions are (yes, you’ve guessed it) attracted techniques: ultrafiltration used impressively small to the cationic resins. The result is that pore sizes, you’ll be even more nicely deionised water comes out of the impressed by reverse osmosis. Reverse other end of the resin bed. 1. Distillation. A technology as old as osmosis filters have pore sizes of less the hills (or at least as the stills that than 0.001 microns, which allows them Commercially available water, or water were hidden in the hills). Water is to sieve ions depending on their purification systems will typically use a heated to its boiling point then diameter. This is used for desalting the combination of these. The higher the water condensed back to liquid. This will water. purity grade, the more techniques used. remove many impurities but impurities

xxxiv (Exactly) Why Enzymes Have Optimal Temperatures

“Actually it’s reassuringly simple...”

Section 10 By Nick Oswald Every biologist is familiar with the Going Up happened, like one of the reactants profile of the rate of an enzymatic became limiting for example. reaction versus temperature as shown in the figure. The first part of the reaction rate profile Plateauing... (shown below in green), where the rate We know that enzymes from E.coli or is increasing with the temperature, warm-blooded tend to have an The reaction rate begins to plateau then fall follows the Arrhenius equation. If the optimum around 37°C while those from in the yellow highlighted section of the enzyme was completely stable even at thermal vent bacteria have much higher graph. This is due to the temperature high temperatures the reaction rate optimal temperatures. approaching the point at which the enzyme would continue to increase with begins to denature (and therefore lose Surprisingly, I find that many biologists temperature until something else activity). don’t have a grasp of why enzymes have these temperature profiles. Actually it’s reassuringly simple.

Chemists have a rule of thumb that a 10°C increase in temperature gives a doubling of the reaction rate. This rule is loosely derived from the Arrhenius equation.

Basically, as the temperature increases so does the kinetic energy of the reactants. This increased kinetic energy means that the reactants are more likely to collide with enough energy to allow the reaction to occur, so the higher the temperature, the higher the A typical plot of enzymes optimal temperature (rate vs temperature). The shape is reaction rate. caused by competition between the rate increasing with temperature (in line with Arrhenius equation) vs the breakdown of the enzyme at higher temperatures. xxxvi Going down

At even higher temperatures (the red section) the enzyme is fully denatured and no activity remains.

The temperature at which the Did you find this useful? denaturation occurs is dependent on the structure of the enzyme, which in turn is related to it’s evolutionary origin. Visit BitesizeBio.com for many more Thus, E.coli enzymes have evolved to eBooks, articles, webinars and videos on cope with temperatures of around 37°C topics just like this one. while enzymes from thermal vent bacteria have been forced to evolve in such a way that they can remain stable at far higher temperatures.

So an enzyme’s optimal temperature is a trade-off between the Arrhenius-type dependence on temperature (the hotter the reaction, the faster the rate) and the instability of the enzyme as it approaches then reaches its denaturation temperature.

xxxvii © 2012 Science Squared Ltd, UK

Photos: Bigstockphoto.com, Sxc.hu

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