PCB 3063L Final Exam Study Guide with all verified answers
.docx
School
Florida State College at Jacksonville**We aren't endorsed by this school
Course
PCB 3063
Subject
Biology
Date
Dec 19, 2024
Pages
10
Uploaded by CommodoreRoseLlama66
PCB 3063L Final Exam Study Guide with all verified answersDo Genomes vary in size? - Yes. Viruses and Plasmids (non-living) have small genomes. Eukaryote genomes are much largerHow do we estimate genomic complexity? - By extracting DNA from an organism, digesting it with a restriction enzyme (EcoR1, Hind III), and running gel electrophoresis.What is Satellite DNA? Where is it common? - In eukaryote genomes, some DNA exists as thousands of repeated sequences. Common around centromeres and telomeres.What was the purpose of Lab #5? - To isolate genomic DNA from Drosophila and analyze the DNA by gel electrophoresis.Explain the procedure for Genomic DNA Isolation of Drosophila - -Grind up 5 flies-Add 200uL SDS Lysis Buffer-Centrifuge to remove debris-Transfer supernatant (clear liquid) to new tube. dispose of solid debris at bottom.-add 200uL phenol:chloroform and vortex-Centrifuge for 4-5 min. Remove 100uL of the top aqeuous layer (DNA, RNA, sugars, salts). Discard middle and bottom layer (Proteins, lipids; phenol waste)-Set aside 5uL of DNA for gel electro. --> label NO RNAse-Add 10uL 10mg/mL RNAse to sample and vortex (+RNAse)-incubate for 30 min.-Set aside 5uL of DNA for gel electro --> label +RNAse-Add 4uL water and 1uL 10x Gel Loading Dye to NO RNAse and +RNAse-Save additional RNAse treated DNA Sample for Lab #6
-TA will run gel electrophoresis on NO RNAse and +RNAseWhat is the purpose of SDS Lysis Buffer? - Is a detergent. Lyses cells (opens them up)What is the purpose of Phenol:Chloroform (CHCl3)? - To remove protein and lipid waste from the genomic DNA ie. purifying nucleic acids. When centrifuged, genomic DNA, salts, and sugars will be in thetop aqueous layer. This layer is immiscible with the bottom organic layer containing the protein/lipid waste.What does RNAse do? - Cleave RNA and removes it from Genomic DNA Sample.What were the results of the gel electrophoresis in Lab #5? - We were Group 3. Both NO RNAse and +RNAse had a distinct marker at the top. NO RNAse had a broad streak at the bottom.Describe Polymerase Chain Reaction (PCR). How many times does the cycle have to be repeated to be visible on a gel? - 1. 94-96°C to denature double stranded-DNA. 4min2. 50-52°C to allow primers to anneal 1 min3. 72°C for extension (amplification) by DNA Polymerase . 2 minDoubles the amount of target DNA. Repeated 25-30 timesQuickly outnumbers original DNA Template to be visible on an ethidium stained bromide gelWhat is Taq Polymerase? - Heat insensitive DNA extracted from bacteria extremophile.What size/length of target DNA can be amplified? - 3k-5k bp.How many bp is the 18S ribosomal RNA (18SrRNA) gene fragment that will be amplified? - 488bp length gene fragment
How many bp is the Actin gene fragment that will be amplified? - 936bp length gene fragmentWhich gene fragment (18SrRNA or Actin) was amplified by our group? - 18srRNAWhich gene fragment (18SrRNA or Actin) will travel further through a gel? - 18SrRNA. It is smaller than Actin (488bp vs 936bp)Describe the procedure of Lab #6- PCR - -dilute 2uL of genomic DNA sample to 38uL water-TA Will add 47.5 uL of Master mix to new tube --->[20um forward primer, 20um backward primer, Taq 5X Master mix- (Taq polymerase, deoxynucleotides, Taq Buffer), 35.5 uL water]-Add 2.5 uL dilute DNA to above. Label tube "DNA"-Add 2.5 uL water to above as control. Label tube "Water"-Run PCR-Set aside 20uL PCR Product for electrophoresis. Save the remaining 30uL for Lab #7Describe molecular cloning - Inserting a DNA Fragment (18srRNA gene fragment from Drosophila) into a plasmid that can be grown in bacteria. Can then transfer to other organisms (eg. human insulin)What is a plasmid? - A small self-replicating, extrachromosomal ring of DNA. Can be grown in bacteria. Usually plasmids carry antibiotic resistance.How to clone a DNA gene fragment: - 1. Isolate gene fragment by PCR2. During PCR Taq Polymerase added "sticky ends"- 3' overhang of a deoxyadenosine (A) to each fragment3. Plasmid vector is prepared to have matching stick ends
4. Plasmid and DNA can then be ligated togetherGeneral Characteristics of Plasmid vectors- to be cloned into bacteria - -engineered to have Multiple Cloning Sites (MCS) --> several restriction sites all clustered together. ---> TOPO© Cloning plasmids have a Topoisomerase I recognition site in MCS-have gene that gives bacteria ampicillin resistance by producing beta-lactamase (cleaves ampicillin)-origin of replication that allows for high replication ratesHow does inserting a plasmid into bacteria make the bacteria antibiotic resistant? - Plasmids have a gene that gives bacteria ampicillin resistance by producing beta-lactamase (cleaves ampicillin)What are important features of the PCR©4-TOPO plasmid vector? - -3956 bp -Ampicillin resistant gene (bla) codes for beta-lactamase-LacZ gene-MCS- genetically engineered part of LacZ gene. lots of restriction sites.How do we know that e.coli has the plasmid? - Ampicillin will kill all bacteria without plasmid (antibiotic resistance via beta lactamase production)What are competent cells? - Cells treated with CaCl2 and heat shock to create pores in membrane/ cell walls.What is the function of DNA topoisomerase I? - cuts and rejoins supercoiled DNA.How does DNA topoisomerase I work? - Covalently binds to phosphate group of 3' end of TOPO plasmid at MCS on each strand (thymine opposite of sticky A). Cleaves phosphodiester backbone and conserves this energy in Tyr-274 residue of Topoisomerase I. Then ligates PCR product into plasmid TOPO vector
How are plasmids inserted into bacteria? - Through transformation of plasmid DNA into e coliDescribe transformation of plasmid DNA into e coli - -Bacteria cells treated with CaCl2 and Heat shock.-Holes pokes in cell walls/membranes. become competent- Plasmid DNA sneaks in- Bacteria spread on plates and treated with ampicillin-only successfully transformed cells will survive and grow.Describe the Lab #7 Procedure - -Add 4 uL PCR Product, 2 uL water, 1 uL salt solution, 1uL Vector (TOPO plasmid containing topoisomerase I)-incubate at RT for 10min-add top 10 cells (bacteria)-ice for 10 min-Heat shock for 30 s at 42°C-ice for 1 min-add 250uL warm SOC to transformed sample. shake 15 min-spread 125 uL on each plate (LB and LB-AMP+)What is the purpose of Bacterial miniprep? - To isolate plasmid DNA from liquid culture (LB AMP+). Ie. remove bacteria and purify plasmid DNA.What is the purpose of Miniprep Buffer P1? - Resuspension of cells in a neutral buffer with RNAse after centrifuging. Made of: Tris-HCl, pH 8.0, EDTA, RNAse.
What is the purpose of Miniprep Buffer P2? - Lysis of Bacterial cells and denaturization of DNA and proteins (eg. alkaline lysis --> releases DNA from bacterial cells)Made of: SDS, NaOHWhat is the purpose of Miniprep Buffer N3? - Neutralizes lysis reaction. Aids in DNA binding to affinity columnMade of: Acetic AcidWhat is the purpose of Miniprep Buffer PE? - Wash proteins, salts and buffers through column, leaves plasmid DNA behind in column.Made of: NaCl, Tris-HCl, EtOH, pH 7.5What is the purpose of Elution Buffer? - Removes (elutes) plasmid DNA from affinity column. Made of: Water.Lab #8 Plasmid Miniprep Procedure - -Transfer 1.5mL liquid bacterial culture (from LB+AMP plate) into tube-Centrifuge. Decant and discard supernatant. Keep bacteria pellet. -Resuspend pellet in 250uL Buffer P1 by vortexing-Add 250uL Buffer P2. Invert gently-Add 350uL Buffer N3. Invert gently-Centrifuge for 10 min-Pipet 850 uL of supernatant to spin column-Centrifuge for 1 min.
-Discard flow through. Wash using 750uL Buffer PE-Centrifuge for 1 min. -Discard flow through. Centrifuge again for 1 min. -Place column in clean microcentrifuge tube-Add 50uL sterile water. Let stand. Centrifuge and discard column. keep flow-through (isolated plasmid DNA)-Electrophoresis (Miniprep Product) 15uL water, 3uL plasmid DNA, 2 uL 10X loading dye. 1.2% agarose.How can the amount of nucleic acid in a sample be determined? - By using a spectrophotometer that emits UV light- DNA and RNA absorb UV light effectively, allowing for quantification even at low concentrationsWhat is the absorption maximum of nitrogenous bases in nucleotides? - 260nmWhat concentrations of nucleic acids of various DNA types (double-stranded, single stranded, oligonucleotides) are necessary for an Optical Density of 1.0 - 50 ng/uL for double-stranded DNA40 ng/uL for single-stranded DNA and RNA20 ng/uL for oligonucleotidesIf the absorption max of nitrogenous bases is 260nm, what does absorbance at 280 nm and 230 nm indicate? - Contamination of sample by proteins and salts.What an A 260:280 ratio of 1.7-1.9 optical density indicate? - A pure sample of Plasmid DNAWhat are restriction enzymes? - Endonucleases that catalyze the cleavage of phosphate bonds within double-stranded DNA. Only cut at 6 base recognition sites (eg. EcoR1 only cuts at 5'-GAATTC-3') Leaves sticky G overhang.
Where was EcoR1 restriction enzyme obtained from? - E. coli strain RY13 (1st enzyme to be isolated)Lab #9 Procedure (Quantifying Plasmid DNA) - -Deposit 1uL sample onto pedestal. -Record A260/280, Concentration (ng/uL-Wipe pedestal with lab wipeLab #9 Procedure (Restriction Digest of Plasmid DNA) - -(+)Cut Tube- 13uL water, 2uL 10X EcoR1 buffer, 4uL miniprep plasmid DNA ( 0.2-1ug DNA... 4uL = 773.6 ng...0.7736ug DNA), 1 uL EcoR1 Enzyme.-(-)UnCut Tube- 14uL water, 2uL 10X EcoR1 buffer, 4uL miniprep plasmid DNA.-Incubate Cut for 1 hour at 37°C-Add 2.2 uL 10X loading dye to each.-Load into 1.2% gel electrophoresis.What is the purpose of Restriction Digestion Analysis? - Determine whether the plasmid DNA Sample acquired an insert (insert should be same size/number bp as PCR fragment). Recognition site for EcoR1 enzyme in vector sequence on either side of insertion.Will result in two pieces of DNA: Plasmid vector and insert fragment (PCR product)Describe DNA Sequencing (Sanger Sequencing). - determine order of nucleotides along fragment of DNAWhat are the components of DNA sequencing? - -DNA Template-An Oligonucleotide primer-Taq polymerase-deoxyribonucleotides-Di-deoxyribonucleotides-Mg++ Buffer
What are dideoxyribonuecleotides? - Analogs of nucleotides lacking a 3' OH group- ie prevents more nucleotides from being added, terminates strand (Chain terminator). Labeled with a fluorescent dye.Describe the process of DNA Sequencing - Essentially a modified PCR reaction (cycle sequencing)-nucleotides incorporated by Taq polymerase as in regular PCR-Di-deoxy chain terminators randomly inserted-Result: series of fragments each ending with different dye/nucleotide and each a unique size.-analyzed by gel electrophoresis (separates based on size)-Fragments are sorted (small to large) and passed through fluorescence detector which identifies dye/nucleotide-Result:ChromatogramDescribe a Chromatogram - A data chart with four different color traces representing the different nucleotides. Peaks to the left are shorter, peaks to the right are longer. Sequence can be read left to right.Lab#10 Procedure (setting up a sequencing reaction) - -Pipet 1.5 uL 300 ng DNA-Add 8.5 uL water-Add 2 uL 1.6 uM primer stock (10ng/uL)-Add 8 uL reaction master mix-Run through thermal cycling.Lab#10 Procedure (Purification of sequencing reaction) - -Add salt buffers to PCR sample (2uL 3M sodium acetate pH 5.2; 2uL 100mM EDTA pH 8.0)-Add 1uL glycogen-Transfer sample (25uL) to new tube
-Add 60uL cold 95% EtOH. Vortex-Centrifuge at 4°C for 15 min. -Decant and discard liquid. Do not disturb pellet-Carefully add 200uL 70% EtOH down side of tube to wash-Centrifuge at 4°C for 2 min-Decant and discard liquid.-Wash again with 70% EtOH. Centrifuge again.-Decant liquid from tube. Let pellet dry for 10 min.-Resuspend pellet in 20uLof 0.1 mM EDTA. VortexWhat is the purpose of Salt Buffers when purifying a sequencing reaction (Lab #10b)? - Salt buffers inhibit any remaining active polymerase. Required for ethanol precipitation of DNAWhat is the purpose of glycogen when purifying a sequencing reaction (Lab #10b)? - Glycogen optimizes precipitation of DNA from solution. Helps to form a visible pellet.