Cell Bio Lab: Final Exam

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SDS-PAGESodium dodecyl sulfate polyacrylamide gel electrophoresis separates proteins based on molecular weight SDS: detergent that causes all the proteins to adopt a negative charge beta-mercaptoethanol denatures the proteins by breaking disulfide bonds separation is based on sizeNative gelnon-denaturing gels separation based on charge to mass ratio, shape, and size2-dimensionalbased on two separation techniques 1st separation using isoelectric point 2nd separation using molecular weight through SDS-PAGEFactors related to gelsVoltage Current Power must remain constant to prevent heat buildup, which distorts or denature the proteinsDiscontinuous Gels2 regions in the gel 1. Stacking gel: lower %T, larger pores, wells to load the protein sample 2. separating/resolving/running gel: higher %T, smaller poresStaining & DestainingCoomassie blue is the general protein stainRfmigration distance of protein from origin (bottom of the well)/ distance the dye front travelled from the originUnits of ProteinsUsually da, Kda, or the proper conversion to g/molStandard Curve for Proteinslog10 of molecular weight (y axis) and Rf (x axis)Cathode (black)negative electrodeAnode (red)positive electrodeWhy did we use Arabidopsis in the Genomic DNA extraction/PCR experiments?Model organism with a small genome and small generation timeWhat part of the Arabidopsis did we use?Inflorescent clustersWas it necessary to use only one part of the Arabidopsis?No, we could use any part of the plant because we were testing for genomic DNA. gDNA can be taken from any portion of the plant because it represents all sequences, such as noncoding or coding, equallyGeneral Procedure of SDS PAGE experiment- Unwrapped gel from kit - secured gel in place with wings - loaded assembly module into the tetra cell tank - removed comb - filled the tank with buffer - loaded about 5 uL of Coomassie blue dye into each wellGeneral Procedure of gDNA Extractions- Prepared buffer - obtained 2 to 3 inflorescent clusters - added gDNA extraction buffer to a tube and used drill and pestle to homogenize the leaves - Added more gDNA extraction buffer - Placed on a heat block - added neutralization buffer and vortexed - Placed on ice - Placed in centrifuge - Removed aqueous layer and dispensed into a new tube - Added isopropanol - Centrifuged - Removed isopropanol without disturbing pellet - pellet washed with ice cold 70% ethanol - centrifuged - poured out supernatant - dried pellet - Added water - stored at 4 C until next labIndication of good DNApresence of a white pelletWhat type of DNA extraction was performed?Crude prep, use of drill and pestle, only testing the presence of DNAIs a crude DNA prep performed in official labs?No, extraction procedure will be more precise to get a higher quality DNA extractionCentral Dogma of Molecular BiologyDNA replication: DNA makes DNA (DNA polymerase) Transcription: DNA into RNA (RNA polymerase 2) Translation: RNA into proteins (mRNA transcript with codons translated by anticodon of tRNA; creation of peptide bonds in ribosome) Reverse transcription: RNA into DNA (use of reverse transcriptase) RNA replication: RNA into RNA (RNA dependent RNA polymerase)Polymerase Chain ReactionAmplification of minute amounts of DNAWho created PCR?Kary Mullis in 1983, won a Nobel Prize in 1993Steps of PCRDenaturation: 45 seconds to 1 minute at 95 C: separates the DNA strands Annealing: 45 seconds at 55 C to attach the primers to each DNA strand Extension:: 1 minute to 1.5 minutes at 72 C to synthesize new strands using free dNTPsPrimers3' hydroxyl groups, which allows dNTPs to be added to the strand must be a forward (5' to 3') primer and a reverse (3' to 5') primer for PCR to take placeWhat is required for PCR to take place?target DNA template Primers PCR buffer (Tris HCl, KCl, Mg2+) dNTPs Taq polymeraseImportance of PCR buffercreates an optimal environment for primer extension by resisting the change in pHWhat is an important characteristic of Taq Polymeraseheat stable DNA polymerase, can withstand the extreme temperature changes in the thermocyclerWhat formula represents the outcome of PCR?2^n, n representing the number of cycles Ex: 4 cycles 2^4 = 16 dsDNA productsWhy do the primers anneal instead of target DNA re-hybridizing??we add primers in excessive molar concentrationWhy does the PCR commonly has 30 cycles?Primers and dNTPs will be consumed, dNTPs are heat-labileWhy the length of PCR products is defined by where the primers are located? Polymerase can ideally extend the products out of the boundary set by primers.Original template runs outDNA quantificationConcentration: Nanodrop A260/A280 = 1.8 (how pure is the DNA)What gene did we amplify?ACTIN gene in Arabidopsis gDNA Forward primer: At-ATCIN-483F Reverse primer: At-ACTIN-D40-R2Why did we multiply the amounts of the PCR content by 4.2x?Since some of the volumes were too small even for a micropipette, we multiplied by 4.2x to make it easier to dispense. Additionally, there were four groups, so we made a class master mix.What was the specific buffer we used for the PCR?5X Green or Colorless GoTaq Reaction BufferWhat was the specific DNA polymerase we used for the PCR?GoTaq G2 DNA PolymeraseAgarose Gel ElectrophoresisHorizontal electrophoresis Concentration of agarose vs pore size (high gel concentration, good for smaller products) Gel strength depends on the concentration Agarose gels have low resolving power compared to polyacrylamide gelsFactors that affect migration of nucleic acidsGel concentration Size of DNA/PCR products Voltage Ionic Strength of the bufferGel concentrationAgarose gel is prepared from the formula of 1% agarose gel = 1g of agarose in 100 ml of TAE bufferSize of DNA/PCR productsDNA is more suitable agarose gel because DNA molecules are usually largerVoltageIf the voltage is too high, it will result in a smiley gel. This results in the melting of the gel, which causes distortion in the PCR band path.Ionic Strength of the bufferUsually use Tris-acetate-EDTA or TAE Also can use Tris-borate-EDTA; TBE dsDNA moves faster in TAE buffer TAE has lower buffer capacity than TBEWhy do we need to stain the agarose gel and what do we use?Since DNA is not visible in natural light, we must stain the gel in Ethidium Bromide or SYBR goldRNA extractionCare needed during RNA extraction RNase free environment RNA more unstable Structural differences in RNA and DNA Tissue/Stage Specific expressionRT-PCRReverse Transcription followed by PCR Use of reverse transcriptase: RNA to cDNA (only exons or protein transcribing genes) Retroviruses, telomerase, retrotransposonsGenomic DNA vs cDNAgDNA expresses all sequences equally, and any part of an organism can be used. It is much larger. cDNA has unequal expressions of the sequences because it is organ specific. It is usually smaller and only has exons.oligo dT vs random primersOligo dt: stretch of 12 to 18 deoxythymine 5' - d( TTT TTT TTT TTT TTT TTT)-3. It used for the production of cDNA from mRNA containing poly A tail. Acts as a primer for the reaction catalyzed by reverse transcriptase. Random primers: mixture of all possible hexmer oligonucleotide sequencesgenomic DNA contamination of RNA samplesimpacts sensitivity and specificity of RT-PCRWhy is a PCR normally done for 30 cycles?Normally PCR is done for 30 cycles because there is a limited number of primers and dNTPs. As the cycles progress, more primers and dNTPs will be consumed to synthesize the DNA strands. Additionally, there is an extreme change in the temperature for PCR, and the dNTPs are heat liable, meaning that they are vulnerable after a certain time of being exposed to the heat.1. In a PCR, what does the annealing temperature depend on? What determines the extension time that should be used during PCR?The annealing temperature depends on the melting point and the length of the primers. The primer melting point is typically determined by the GC content. For example, the forward primer (At-ACTIN1-483 F) had a melting point of 66 °C and a GC content of 50%. The reverse primer (At-ACTIN1-D40-R2) had a melting point of 66 °C and a GC content of 57%. The extension time depends on the length of the primers, and the extension time is set during cycle 2 when the newly synthesized strands are constrained by the primers. Additionally, the primer extension depends on how quickly the selected DNA polymerase can synthesize new strands. While Taq polymerase is used in this PCR, other heat stable polymerases can synthesize faster or slower depending on their characteristics.When designed primers, what should you keep in mind?They should be at least 20 to 21 nucleotides in length for proper specificityHow can you estimate the size of amplified PCR product during gel electrophoresis?The size of the amplified PCR product can be estimated by using a DNA ladder, which gives scientists an already known baseline lengths of the DNA. Using these predetermined lengths, scientists can estimate the length of the DNA bands depending on where they reside in the gel.How long was the DNA ladder that we used?1000 b (1 kb) longHow long was the expected PCR product?It was 806 bp, but it was estimated between 800 and 850 bp using the gel in the lab manual.