Molecular Methods definitions lectures 7-10

What are the two types of spectrophotometer?

1. conventional single beam spectrophotometer (Shimadzu UV-1200) 2. Diode array spectrophotometer (Hewlett-Packard HP 8452A, Agilent 8453)

Proteinase K what is that?

Proteinase K (that can also be used in the "removing contaminated proteins in the lysate" step of isolating total cellular DNA LIKE Quigen protease) is what Quigen protease is an alternative for. Proteinase K is a subtilisin type serine protease from the fungus Tritirachium album. (Subtilisin is a non-specific bacterial protease family from Bacillus--Quigen protease is from a recombinant Bacillus remember?) Proteinase K has a molecular mass of 28.9 kDa and it cleaves at hydrophobic aliphatic and aromatic AA residues (whereas Quigen protease cleaves at neutral and acidic AA residues)--this is a difference between the two but still they both get the job done and eat the damn proteins Next, Proteinase K is stable over a WIDE TEMPERATURE RANGE of 37-60 degrees C and pH from 4-12.5 (so acidic to basic)--this means that Proteinase K is NOT inhibited by EDTA Calcium is NOT REQUIRED for enzymatic activity of Proteinase K

Recall the first three buffers for plasmid isolation

R3 (with EDTA and RNase A), L7 (with SDS and NaOH), and N3

Purification of DNA solution with organic solvents targets the removal of what?

removal of proteins

After completion of the CsCl plasmid purification method, how can the supercoiled plasmid DNA be extracted, as well as the removal of the CsCl and extraction of the EtBr?

- can remove the supercoiled plasmid DNA with a syringe - remove the cesium chloride (CsCl) by dialysis where the CsCl will ultimately diffuse into the buffer - extract the ethidium bromide (EtBr) with n-butanol, it will put EtBr in the organic phase and keep the DNA in the aqueous layer

What are the three organic solvents that are used for purification of DNA for protein removal?

1. Phenol-this organic solvent is neutral or slightly alkaline (pH8); Recall that DNA is negatively charged, so in this phenol solvent, DNA is going to be less soluble 2. Phenol/CHCl3- volume ratio of 1:1- this solvent is more effective in removing proteins. The CHCl3 part of the solvent removes phenol from the aqueous layer (phenol is partially soluble in water) 3. Phenol/CHCl3/Isoamyl alcohol- volume ratio of 25:24:1- Isoamyl alcohol is used to reduce foaming during extraction (by now you know characteristics of Phenol and what CHCl3 does (removes phenol from AQ layer)

What two methods can be used for isolating total RNA in a cell?

1. The detergent and phenol method: This method uses phenol, strong detergent like TPNS or Tri-isoPropylNaphthalene Sulfonate, and a chelating agent like sodium 4-aminosalicylate 2. Guanidium methods: These methods use a high concentration of guanidium to lyse cells and inactivate RNase (a stronger protein denaturant than phenol). There are a few types of guanidium methods for total RNA isolation: - guanidium acid--phenol method - non-phenol-based (column based) method using the Qiagen RNeasy kit - Guanidium--cesium chloride cusion method

Now getting into talking about the Quantitation of DNA (the plasmid, and the PCR product), as well as quantitation of RNA...there are five methods for quantitation. What are they?

1. The first method for quantitation is the spectrophotometric method that utilizes UV light absorption. 2. diphenylamine method- for quantitation 3. EtBr (ethidium bromide) fluorescence of DNA--this method is a rapid SEMI-quantitative method 4. Hoechst 33258 (fluorescent dye) method for quantitation 5. PicoGreen (OliGreen, RiboGreen, SybrGreen) dye based fluorescent assay method

What are the four forms that plasmid DNA can take?

1. covalently closed circular DNA (CCC, Supercoiled) as was the plasmid DNA used in the CsCl purificaiton method 2. open-circular (OC) (relaxed or nicked) 3. linear 4. denatured

What are the two advantages of diode array spectrophotometer?

1. the diode array spectrophotometer is high speed, spectrum is acquired in 100 msec (fixed grating and hundreds of detectors) 2. the diode array allows entire UV-VIS spectrum to be obtained using polychromatic light

Advantages of CsCl plasmid purification method with density gradient centrifugation name the disadvantages too

1. you get a high yield and purity (A) 2. it's easy to scale up (A) 1. ultracentrifugation takes a long time (40 hours) so CsCl method is super time-consuming (D) 2. CsCl itself is expensive (D) So CsCl plasmid purification method is very time-consuming and expensive but it gives good results

There are three ways to purify plasmids. What are the three ways?

3 ways of plasmid purification: 1. Cesium chloride (CsCl) equilibrium buoyant density gradient centrifugation or "isopycnic centrifugation" (this purification method can be done later on in the plasmid isolation process once you have centrifuged the supernatant plasmid and resuspended the DNA pellet in 70% ethanol and TE buffer to obtain the crude plasma DNA solution in the TE buffer. From here the isolation process can either continue, or this solution can undergo CsCl density gradient centrifugation purification method) 2. Anon exchange column chromatography (this purification method can be used right after you have treated the cells with the three buffers discussed previously (R3 L7 and N3) to obtain that supernatant plasmid--it is this supernatant plasmid that can now undergo anion exchange column chromotography for purification) 3. Glass milk/silica gel method

What are three types of light sources that can be used in a conventional spectrophotometer?

A light source is another major component of a conventional spectrophotometer. There are three different types of light sources that are used for different ranges of light. For example, the Tungsten-halogen lamp is used from 340 to 1100nm, so the Tungsten-halogen lamp is used in the VISIBLE region. The Deuterium lamp is used from 200 to 800 nm, also qualifying the lamp for the VISIBLE range but also for the UV region as well. Last is the Xenon lamp ranging from 220 to 700 nm, again able to work in both UV and visible regions. So recap: 1. Tungsten-halogen lamp: 340-1100nm--visible light only 2. Deuterium lamp: 200-800 nm--both visible and UV light 3. Xenon lamp: 220-700nm--both visible and UV light

What is a monochromator?

A monochromator is one of the major components of a conventional spectrophotometer (PLOSSM). It is used to select monochromatic light or light with a specific wavelength. Monochromators use a prism or a diffraction grating to generate desired wavelengths

DEPC stands for? What is it?

DiEthylPryoCarbonate is what it stands for and diethylpyrocarbonate (DEPC) is an alkylating agent that inactivates nucleases by alkylation. DEPC is typically used to treat water and solutions before working with RNA that is easily degradable. (this makes sense) Traces of diethylpyrocarbonate (DEPC) can actually modify purine residues (A&G) in RNA by a process called carboxymethylation. So because DEPC can modify these RNA purine residues, the DEPC must always be removed from solutions or containers by autoclaving (high P and T). Heating by autoclaving decomposes DEPC to CO2 and ethanol.

What are the next three buffers used in plasmid isolation after R3, L7, and N3?

EQ1- equilibrium buffer W8- wash buffer E4- elution buffer

Where on the absorption spectrum does ethidium bromide alone absorb UV light?

EtBr has absorption maxima at 210 and 285 nm.

Where does EtBr-DNA complex absorb UV light?

EtBr-DNA complex absorbs UV light at 590 nm. It also emits orange-red fluorescence at 590 nm. It should be noted that the fluorescence is 20X stronger than that in the absence of DNA due to a hydrophobic environment.

Walk me through what happens with the diode array spectrophotometer

First the polychromatic light passes through the sample cuvette via a series of lens, shutter, and slit and is then focused on a grating (the polychromator). Then the polychromatic light is dispersed into different wavelength by the polychromator and then is focused on the diode array (which are linear arrays)--a series of photodiodes up to 1,024 of them! No waaaay. The diodes absorb the light and generate a current which is proportional to the numbers of the photons. Each diode measures a portion of spectrum; therefrore, since there are diodes all over the ****ing place, light of ALL wavelengths is measured SIMULTANEOUSLY on the diode array. Data is acquired in parallel. The relative positions of sample and grating are reversed. Has a "Reversed Optics" configuration when compared to conventional

Now moving on from plasmid isolation and purification...let's now turn our attention to isolating TOTAL CELLULAR DNA (nuclear, mitochondrial and chloroplast DNA). How does one go about this?

For isolating total cellular DNA, you first need to have disruption by homogenization (so grinding up the solution with a blender, mortar and pestle, polytron). Then perform the lysis of the homogenate and cultured cells. Remove the contaminated proteins in the lysate by protease (it will digest the proteins) Recover the DNA by removing the RNA contaminants and you'll have the total cellular DNA

Describe the boiling method for plasmid isolation because all we've talked about up until now was the alkaline detergent lysis method using the PureLink HiPure Plasmid Midi Kit

In the boiling method for plasmid isolation, the cells are mixed with TE buffer containing lysozyme and RNase A. The lysozyme is used for digesting cell walls. The cell solution is then boiled to disrupt the cell walls and denature the proteins and DNA both chromosomal AND plasmid. After cooling, the plasmid DNA renatures and remains in solution (sounding familiar?) but the chromosomal DNA only renatures partially, eventually forming a precipitate which is then removed via centrifugation (this is sounding REALLY familiar) This boiling method, UNLIKE the alkaline lysis detergent method, is super quick for preparing plasmids from large numbers of cultures; HOWEVER the DNA quality is much lower than the detergent method. And this is why we used the detergent method in lab...I get it now.

question concerning the last 3 buffers used in the plasmid isolation kit: Which buffer contains the highest concentration of NaCl?

Last 3 buffers used in plasmid isolation kit in order are EQ1 equilibrium buffer (NaCl 0.6M), then W8 wash buffer (NaCl 0.825M), and lastly E4 elution buffer with (NaCl 1.25). Therefore, the E4 elution buffer has the highest salt (NaCl) concentration at 1.25M.

Describe some characteristics of phenol and why it's such a good agent for purifying DNA--describe phenol extraction

Phenol extraction is used to purify samples of NA taken from cells. Phenol is so effective because for one, phenol is a NON-POLAR compound. NA on the other hand are polar. Therefore, nucleic acids DO NOT dissolve in the presence of phenol!!! Also, phenol has a density higher than water's. So when phenol is added to a solution and centrifuged, you get two phases or layers: an aqueous layer (always on the top) and an organic layer (on the bottom). The aqueous phase is a polar phase; therefore, it will contain the DNA and RNA nucleic acids with water. The organic phase (the denser phase on the bottom) will therefore contain the denatured proteins, the phenol and the other cell components. This is KEY in understanding why we do Phenol extraction.

What on earth is Qiagen protease?

Qiagen protease, although sounding like the name of a Jedi, is actually a serine protease isolated from a recombinant Bacillus strain that cleaves at neutral and acidic amino acid residues--NOT BASIC ones Qiagen protease is also an economical alternative to proteinase K For Quigen protease to have full activity, the EDTA must be less than 8mM (if it's more, then the protease will lose its activity) Just on a note pH of EDTA is around 8 which is slightly basic so this might have something to do with inhibiting Quigen protease's function if there's too much EDTA--just a thought also note that Qiagen protease is a protease that can be used in the "removing contaminated proteins in the lysate" step of isolating total cellular DNA (there's your application)

What are slits?

Slits are another major component of the conventional spectrophotometer (ploSsm). Located IN the Optical system. Slits are used to determine the intensity of light and spectral purity (makes sense because this is a function of the Optical system as well which contains Slits); Slits are controlled by a computer

characteristics of chaotropic ions/compounds

So chaotropic ions are large, monovalent ions with LOW charge densities. These compounds when in high concentration will denature macromolecules like proteins, DNA, RNA, and they will also remove water from hydrated molecules that are in solution (so the DNA and the silica gel)

Describe the Guanidium acid--phenol method of the Guanidium methods for total RNA isolation

So in the Guanidium acid--phenol method for total RNA isolation, the cells are first lysed with 6M (a high concentration) of GuHCl (or guanidine hydrochloride) Then it is centrifuged, leaving the nucleic acids (both DNA and RNA) in the supernatant which you will then precipitate out with 3/4 volumes of 96% ethanol. The Nucleic acid pellet that precipitated will then be suspended in TEN-SDS (slightly alkaline) and then you will extract the NA with acidic phenol/CHCl3 (one of the organic solvents that is more effective in removing proteins. The CHCl3 part of the solvent removes phenol from the aqueous layer, because phenol is dense and moves to the organic (bottom) layer with the other organic contaminants; the NA are in this aqueous layer where the polar molecules are)--so: NA is extracted out into the aqueous layer. Then you add one drop of HAc, NaCl to 0.2 M, and you add 6/10 volumes of isopropanol to the aqueous layer containing those nucleic acids, to precipitate the nucleic acids (NA). So now you have the DNA and RNA only. Next, suspend those NA in diethylpyrocarbonate-treated water and add MgCl2 to 1mM for RNA aggregation and LiCl to stock; incubate the sample at 4 degrees C overnight or -20 degrees C for at least 2 hours. The significance of this incubation is to get RNA to precipitate out while the DNA remains soluble. The large RNAs like rRNA, hnRNA, and mRNA will precipitate but the small ones (5S rRNA, 5.8S rRNA, and tRNAs will be stubborn and remain soluble with the DNA. So then we will have to centrifuge the remaining solution with the DNA and the small RNAs to get an RNA pellet which we will then dissolve in water and re-extract with TE saturated phenol--The water and the rest of the RNAs will form the aqueous layer. This aqueous layer will then be precipitated out with 2 volumes of 96% ethanol with 0.3 M NaAc at a pH of 6 to get the final components of total RNA isolation.

Describe the Glass Milk/ Silica gel method for plasmid purification

So in this method, the plasmid DNA is mixed with glass milk/silica gel in the presence of a high concentration of chaotropic compounds (A chaotropic compound denature macromolecules and remove water from hydrated molecules in solution) This in turn leads to the dehydration of both the silica surface and the DNA, allowing the DNA to hydrophobically bind to the silica resin, being absorbed to the silica at a low pH (7.5 or less). Then after this absorption of the dehydrated DNA to the dehydrated silica gel, the silica gel is washed with salt/buffer to remove any impurities and chaotropic compounds. Finally, the DNA is eluted with a LOW SALT BUFFER such as 10mM Tris-HCl, pH8.5 or water. so in a nutshell for this silica gel (glass milk) membrane plasmid purification process, a high salt concentration is used to bind the DNA to the silica gel (this is where the chaotropic compounds come into play), but the elution is done with low salt. The eluate (substance obtained by elution--so in this case the purified plasmid) is ready to use. Microspin colums are used and there are mini preparations

side question: why does the DNA interract with the DEAE resins?

So the DEAE resins on the silica gel are positively charged and they therefore interact with the negatively charged groups of the DNA molecules.

Now describe the last of the three Guanidium methods for total RNA isolation: The Guanidium--cesium chloride cushion method

So this Guanidium--cesium chloride cushion method for total RNA isolation is a time consuming method and only a limited number of RNA samples can be isolated. Guanidium ISOthiocyanate is used to lyse the cells and simultaneously inactivate any ribonucleases. The lysate then undergoes ultracentrifugation (Cesium chloride density gradient centrifugation) through a 1.2M and 5.7M CsCl cusion--hence the name of this total RNA isolation method) The RNA, which is denser than the DNA and protein, will pellet out at the bottom of the tube; however, small RNAs cannot be prepared because they are not recovered. (referencing back to the fact that this Guanidium--CsCl cushion total RNA isolation method relaly only lets a limited numer of RNA samples be isolate)---so is it really a total RNA isolation method though...? Really......

Concerning isolating total cellular DNA, walk me through preparing eukaryotic DNA by phenol extration

So you're going to suspend the cell pellet in buffer I containing EDTA, which will form a chelate with divalent ions, weakening cell walls (EDTA in the buffer will ultimately weaken the cell walls). Then proteinase K (the enzyme used in the removal of contaminated proteins step of DNA isolation) will be added with SDS and the sample will be incubated for an hour. Following incubation, DEPC or diethylpyrocarbonate will be added to the sample and the sample will have to be incubated again at 70 degrees C for just 15 minutes this time. Following this second short incubation, 5 M KAc will be added and the sample will be put on ice for 30 minutes. The significance of putting the sample on ice upon KAc addition is that it will allow the proteins, digested by Proteinase K, as well as the SDS to precipitate out of solution into a pellet. Then the remaining portion of the sample will be centrifuged-->the aqueous layer will be extracted with TE-saturated phenol (pH8--slightly alkaline) and then it will eventually be precipitated out by addition of 2 volumes of 96% ethanol. This DNA pellet will then be washed with 70% ethanol, vacuum dried, and suspended in TE pH 7.6 (neutral) or just water. (And so we have extracted total cellular DNA by using phenol to ultimately extract out the DNA-containing aqueous layer to later on be precipitated out into a DNA pellet.)

Describe the Cesium Chloride plasmid purification method

The CsCl method is a type of density gradient centrifugation for plasmid purification. So what happens is the crude plasmid DNA in TE buffer solution from the plasmid isolation process is then mixed with solid CsCl (cesium chloride) (0.95 gm/ml) and ethidium bromide (final conc is 0.4 mg/ml). After mixing this solution, the DNA solution is subjected to ultracentrifugation (that is the density gradient centrifugation) and remember from earlier on that rotors used for density gradient centrifugation are usually either Vertical C or Swinging bucket B rotors. For the CsCl method, a swinging bucket B rotor is used for the ultracentrifugation step at 100,000-140,000 xg for 40 HOURS so at least overnight. Upon this ultracentrifugation, the chromosomal DNA binds more ethidium bromide (EtBr) than the supercoiled plasmid DNA. (here it is important to note that this DNA solution used in the CsCl plasmid purification method STILL CONTAINS BOTH DNA FOR THE PLASMID AND FOR THE HOST CHROMOSOME--although most of the host chromosomal DNA should have precipitated out with the SDS/lipid/protein pellet formed from the treatment with all those buffers (R3 L7 and N3) earlier in the plasmid isolation process. But anyway, that chromosomal DNA binds more EtBr (density 0.34) than the supercoiled plasmid DNA, so it can be inferred that the chromosomal DNA has a lower molecular density than the plasmid DNA. This means that the plasmid DNA is going to sink more towards the bottom of the tube where there is more CsCl. Also, just another note: supercoiled plasmid DNA is topologically constrained (not easily unwound) with its covalently-closed circular (CCC, supercoiled) structure, so it binds less EtBr and again has a higher molecular density than the chromosomal DNA So basically...with CsCl method, the plasmid DNA is shown to be lower in the tube, binding EtBr less, and having a higher molecular density than the chromosomal DNA. Any RNA that was in the crude DNA solution will be all the way at the bottom of the tube, easily distinguishable

Concerning quantitative PCR, define Ct (Cp) value

The Ct (threshold cycle) is defined as the number of cycles required for the flourescent signal to cross the threshold (i.e. exceeds background level) Ct values are inversely proportional to the amount of target template DNA in the sample (meaning the lower the Ct level, the GREATER the amount of target DNA in the sample)--INVERSE RELATIONSHIP

What is the optical system?

The Optical system is another major component of a conventional spectrophotometer (plOssm). The Optical system contains Slits, lenses, filters, and mirrors. Notice Slits is also one of the six major components of a conventional spectrophotometer (ploSsm), and slits are in the Optical system. The Optical system is used to concentrate light, increase spectral purity, and focus light toward the sample

Describe what happens with a PureLink HiPure Plasmid Midi Kit

The PureLink HiPure Plasmid Midi Kit references back to the plasmid isolation process. It comes with a DNA-binding column that is packed with DEAE resin. This is the thing that the cells were put into at the beginning of the plasmid isolation process and added all those buffers (R3, L7, N3) to it. So the next step still using this thing is to equilibriate the DEAE with EQ1 equilibrium buffer. Next, use the filtration cartridge to remove the white insoluble precipitate. (This precipitate should contain the SDS/lipid/protein/chromosome stuff that formed from adding the first few buffers R3, L7, and N3). The plasmid will be bound to the DEAE-resin in this DNA-binding column. Now, you add the wash buffer (W8) to remove any impurities such as proteins, metabolites, RNA, and other low MW molecules-->so now you have this ultrapure plasmid DNA which is lastly going to be eluted with elution buffer (E4), a high salt buffer with pH 8.5 (slightly alkaline)

Genomic DNA can also be prepared with a Quigen genomic DNA isolation kit. What is the significance of this?

The Quigen genomic DNA isolation kit does not use phenol and chloroform; it does anion-exchange chromotography. Sample in column-->lyse cells and digest with protease, bind genomic DNA to column, wash, elute-->isopropanol precipitate-->pure genomic DNA is the result

What is a sample chamber?

The Sample chamber is another major component of a conventional spectrophotometer (plosSm). The Sample chamber is used to HOLD a cuvette or sample (thus "chamber") There are three different kinds of cuvettes used for conventional spectrophotometers, so the sample chamber can hold any one of those three kinds of cuvettes. The three kinds of cuvettes are Glass, Quartz, and Polystryrene

What is a phototube?

The phototube is a major component of the conventional spectrophotometer (Plossm), and it is used to detect light intensity and the phototube also converts light energy into an electrical signal which is then red into a recorder.

There are 6 major components of a conventional spectrophotometer. What are they?

The six major components of a conventional spectrophotometer is the light source, the monochromator, the optical system, slits, sample chamber, and the phototube. Think PLOSSM (Phototube, Light source, Optical system, Slits, Sample chamber, Monochromator). PLOSSM for the 6 major components of a conventional spectrophotometer. PLOSSM

What are the three different kinds of cuvettes that can be used in the Sample chamber of the spectrophotometer and describe each one.

The three different kinds of cuvettes used in spectrophotometers are: 1. Glass cuvette- used for visible regions (AKA above 340nm) --*connection: so for visible region only the light source used could be the Tungsten-halogen lamp (340-1100nm)* could also use the other two if range fell anywhere from 340 to 700 or 800 2. Quartz cuvette- used for both UV and visible regions (200-800nm) --*connection: so for both visible and UV regions specifically with a range of 200 to 800 nm the perfect light source to use would be the Deuterium lamp* 3. Polystryrene cuvette- used for only visible regions (350-800 nm) --so for this one could use all three (except Xenon lamp only goes to 700)--but other than that it don't matta

Now we have walked through total RNA isolation with specifically, the Guanidium acid--phenol method of the Guanidium methods for total RNA isolation. An alternative of the Guanidium methods is isolation and purification of total RNA with the Qiagen RNeasy mini kit. Describe that method.

This Guanidium method of total RNA isolation is NOT phenol-based, rather, it's column based. So with the Qiagen RNeasy mini kit the cells are lysed with high concentrations of RLT or guanidine thiocyanate and then homogenized. Ethanol is then added to the lysate solution, giving a final concentration of 35%. This lysate is then loaded onto the RNeasy midi column, ONLY RNA BINDS TO THE SILICA GEL MEMBRANE (recall we also used silica gel in the glass milk method for plasmid purification--connection!) After RNA binds the silica gel, all contaminants (what's not bound to the column essentially) are efficienctly washed away with RW1 and RPE (77% ethanol). Then all that's left--the pure, concentrated RNA (>200 nt) is eluted in water. Done

How can one isolate poly(A) containing RNA in eukaryotic mRNAs?

You can isolate poly(A)-containing RNA in eukaryotic mRNAs with isolation by affinity chromotography The column will contain oligo(dT)-cellulose OR poly(U)-sepharose (so basically either 10-20 of dT or U)--but both of those are complementary to base A either way. So the purification involves RNA binding (poly(A) to either dT or U--whatever is in the column), washing out contaminants, and finally, eluting out that poly(A) containing RNA.

What are the three types of Guanidium methods used for isolating total RNA in a cell?

guanidium acid--phenol method - non-phenol-based (column based) method using the Qiagen RNeasy kit - Guanidium--cesium chloride cusion method

Contrast anion exchange chromatography plasmid purification method with the silica gel membrane/glass milk method

silica gel membrane method uses a high salt concentration to get DNA to bind silica gel; whereas, anion exchange uses low salt to get DNA to bind the column. Silica gel membrane method uses a low salt concentration to elute out the DNA; whereas, anion exchange uses a high salt concentration to elute. With the silica gel membrane method, the eluate is ready to use; whereas, in anion exchange the eluate must first undergo alcohol precipitation. Lastly, silica gel membrane method uses microspin columns and does mini preps; whereas, anion exchange uses gravity flow columns and does midi and maxi preparations.

examples of chaotropic compounds or ions

there are 4 examples of chaotropic compounds 1. sodium iodide (NaI) 2. Guanidine HCl 3. Guanidine thiocyanate 4. Urea