Practical 5 - PCR & Restriction Enzymes & Gel Electrophoresis Worksheet

Task A: Polymerase Chain Reaction Master Mix Your first task is to isolate and amplify the NRAS gene from cDNA extracted from different samples through PCR. You will need to run a total of 7 PCRs: 3 normal samples, 3 cancerous samples, and 1 negative control. To make things easier in the lab, when running multiple reactions, the components are prepared not individually, but as a master mix—all the components for multiple reactions are prepared in bulk, except for the template DNA, which is added separately once the master mix has been distributed into individual tubes. The table below lists the different components for PCR, the available stock concentrations of these components, and the needed working concentrations for the PCR itself. Complete the table by supplying the needed volumes of each component for a single reaction and for the master mix (the first row has been filled in as an example).

TOPIC: PCR and Gene Cloning Basics Question: What are 2 possible roles of CaCl2 in the transformation process?

ersonal use only, do not reple 2021-05-12 1ore 7. Restriction endonucleases recognize particular sequences of DNA and then cut the 16@gmail.com DNA into fragments (see table below). RESTRICTION ENDONUCLEASES BamHI DNA SEQUENCE RECOGNIZED* Luse only, do no repro 2021-05 va16@gmail.com GIG ATC C С СТАGJG GỊA A TT C СТТААЈG Pers EcoRI * Į indicates where the DNA backbone is cut The base sequences of DNA from two different individuals, Alice and Ben, are given below. Indicate where BamHI and EcoRI would cut these DNA sequences by drawing lines (for hints refer to FIGURE 2 and the video in the Canvas assignment for this section ). Personal use ofy 2021-05-12 forevermayal6@gmail.com BEN LA ALICE G C G C uCE G C Personal use only. do no G C 2021-05-12 A T Com G C G C forevermaya16@gnT A C G A T ТА C G C G C G G C G C А Т PersA T G C vermaya16@amail.com T A use only, do not reproduce. 2021-05-12 A T А Т А Т ТА ТА ТА C G 7. Does BamHI cut the DNA from Alice and Ben in the same place? C G…

Incorrect 1 A Help Center? It can sequence a whole genome in one huge strand Each nucleotide has a different marker attached Question 11 LO62 Explain how SANGER sequencing works Order the steps that are needed for gene sequencing through Sanger technology: Amplify DNA Cut DNA into small pieces Add DNA to be sequenced + DNA polymerase + dNTPs + ddNTPs into different test tubes Run resulting sequences through gel electrophoresis 3 111 0/1 pts 0 / 1 pts

A Moving to the next question prevents changes to this answer. Question 1 Which step is NOT included in the technique called Southern blotting? O a. visualization by autoradiography or fluorescence imaging Ob. exposure to a 32p-labeled DNA probe Oc amplification of the restriction fragment-probe duplex d. hybridization of the probe with a restriction fragment having a complementary sequence Oe transfer of the mixture of restriction fragments into a membrane composed of nitrocellulose &Moving to the next question prevents changes to this answer. MacBook Pro esc Q Search Secure Search %23 2 & LO % 4

A sunyocc.open.suny.edu/webapps/assessment/take/launch.jsp?course_assess * Question Completion Status: QUESTION 2 Correctly match the material/supply to its role (job) in genetic engineering. A. vector cuts gene and recipient DNA leaving sticky ends B. DNA ligase v moves newly combined DNA from a test tube into the organism C. restriction enzyme v living thing that gets a new gene and therefore a new trait D. host v glues the new gene and recipient DNA together QUESTION 3 Which of the following are PROS (good things) about genetic engineering? Choose ALL that apply. OA. better health or quality of life B. more products which means lower costs O C. animal welfare concerns O D. more toxins in the environment O E. safer method of sensing explosives OF. more herbicide in the environment O G. fewer crop varieties O H. cool pets QUESTION 4 Which of the following is FALSE about biotechnologies? Click Save and Submit to save and submit. Click Save All Answers to save all answers.

You need to complete the following steps for an experiment. Explain whether an acrylamide gel or an agarose gel is more appropriate to use for the following experiments. Purifying tRNA from total RNA Isolating a vector insert for cloning Analyzing PCR products

at Arrange Window Help 4) 57% D Tue 4 May Tools Slide Show 2 ARMS PCR nations Slide Show Review View A A =、=<|EE|三v| v v A Sha v 20 Quick Styles D Sha Text Arrange Convert to SmartArt Picture Shapes ab x x. AV v Aa v D. Av A、申。 Box Briefly outline how MLPA probe-sets can simultaneously detect multiple targets using a single set of PCR primers. E Notes Comments MAY 4 Pr w MacBook Air

Please answer fast 1. Genome annotation refers to all of the following except a. assigning a unique identifier given to a DNA or protein sequence recordb. locating genes in sequencesc. adding known functions to the sequenced. assigning the sequence to a functional categorye. All of the above are correct 2. Which of the following databases contain curated annotations about human genes? a. GENCODEb. RefSeq c. UniProtKBd. All of the above 3. Which database covers a wider range of functional definitions and therefore would bea better choice for exploring the genome for new genes? a. GENCODEb. RefSeqc. UniProtKBd. All of the above 4. AKT1 is the official ___________________ for a gene. a. gene IDb. namec. symbold. all of the above 5. Which BLAST (Basic Local Alignment Search Tool) variant would you select toconduct a translated nucleotide query? a. BLASTnb. BLASTpc. BLASTxd. TBLASTne. TBLASTx

BIOTECHNOLGY Date: Name: Instructor: Section/Group:. POST-LAB QUESTIONS 1. In one or two sentences, summarize the technique of gel electrophoresis. 2. How does the process of gel electrophoresis separate DNA fragments? 3. Why is the fact that DNA has a negative charge so important in the gel electrophoresis process? Biotechnology 165

Answer: Task 2: Polymerase Chain Reaction After purifying the gDNA extract, you want to isolate and amplify the polystyrenase gene. You perform PCR using the appropriate gene-targeted primers. What is the purpose of the following PCR components? Answer briefly but completely. DNA polymerase isolated from Thermus aquaticus Answer: a. b. Deoxynucleotide triphosphates (dNTPs) Answer: Forward and reverse primers Answer: с. Task 3: Agarose Gel Electrophoresis Your amplicon from PCR was subjected to AGE for analysis. You are shown the image of the gel loaded with the following lanes: (A) negative control, (B) size ladder (1, 2, 3, 4, 6, and 10 kb), (C) GDNA extract, (D) PCR amplicon. However, due to mishaps while loading the gel with the samples, you are not sure which lane is which. You are shown a diagram of the obtained gel below. a. Label each lane of the gel. Write only the corresponding letters in the wells above. b. Above each band in the size ladder, write its size (in kb). c.…

Copy and paste the link below and watch the video on Youtube and Answer the Questionshttps://www.youtube.com/watch?v=g-dNJdOvBM4 Polymerase Chain Reaction  Questions: 1. What are the materials used for the polymerase chain reaction?  2. Draw a schematic diagram of the procedure in PCR.  3. Why is it important to design the primers at the start of the laboratory procedure? 4. What are the components of the PCR buffer and what is its pH range? What is the purpose of the buffer? 5. What is the use for magnesium chloride?  6. How much template DNA is added? What is the concentration of the primers? 7. At what temperatures does denaturation, annealing and extension occur? Why are the processes placed in that temperature? 8. In this particular PCR experiment, how many cycles was used? 9. Can this PCR be used on its own to find out if a person has Covid or not on its own? Why or why not?

O e. Gene amplification CLEAR MY CHOICE To study the function of any gene of interest you would perform the loss and gain of function approaches by either deleting or re-expressing the gene of interest, which of the following can be used to determine and quantify the activity of the gene? Oa. Western blotting O b. Gene knockdown O. PCR/OLA O d. Microscopy O e. DNA hybridization Paternity testing can be detected most precisely by using technique

DNA Restriction, Electrophoresis 1. What are restriction enzymes? 2. How do restriction enzymes function? 3. Why are restriction enzymes important in biochemistry labs? 4. What is DNA Gel Electrophoresis? 5. How does DNA Gel Electrophoresis function? 6. Why is DNA Gel Electrophoresis important in biochemistry labs?

Q1 Homework • Unanswered Q1. I want to do a PCR experiment that requires a final concentration of 2 uM of primers. If my primer stock solution is 10 uM, and my reaction volume is 25 ul, how much primer solution do I add? Select an answer and submit. For keyboard navigation, use the up/down arrow keys to select an answer. a 1 ul 2.5 ul 5 ul d 10 ul 5.

Section Name MAPPING PRACTICE #4 Plasmid pBR 607 is a 2.6 Kb plasmid containing Ampicillin and Tetracycline resistance markers, an origin of replication, and unique restriction sites for the restriction enzymes EcoRI, BamHI, and Pstl. Given the restriction map for pBR 607 for the enzymes EcoRI, BamHI, and Pstl, show on the gel diagram, where the approximate positions of the restriction fragments generated from the restriction digests would be located after carrying out electrophoresis. BamHI 0.2 Kb pBR 607 ECORI 1.94 Kb 0.46 Kb Pstl Size EcoRI EcoRI EcoRI + Standards BamHI + Pstl BamHI Pstl 4.0 Kb 2.2 Kb 2.0 Kb 0.5 Kb

Horizontal sequence :RIVL Vertical sequence:FMK   Scoring rules: g/o = -3, g/e = -1, match or mismatch - from PAM250 substitution matrix below.  NW algorithm.    1. Complete the scoring matrix.    Scoring matrix with PAM250 scores:   R I V L F         M         K           2. Set up, initialize and complete the NW matrix.  3. Retrace, align and score alignment(s).             Use the arrows and circles for the matrix and path(s).                                                                                                                                                                                                                                                                                                                                 R I V L             F           M           K           Align and score all optimal alignments here.  PLZ  the arrows and circles for the matrix and path(s) AND SHOW ALL possible Alignment  Here the following…

I want to know how can I make RNA transcript through next dna sequencing. 5'ATGATCTTTAAAGGGCCC 3'

Question 23 Match the following events in the recombinant DNA and cloning process Prompts gene of interest and the DNA vector are separately cleaved to generate ends that can join DNA is introduced to host cells vector DNA and the gene of interest are joined together only the clones with the recombinant DNA are used for downstream applications Submitted Answers Choose a match transformation marker-assisted selection directional cloning ligation by a ligase action restriction enzyme cleavage

Horizontal sequence :RIVL Vertical sequence:FMK   Scoring rules: g/o = -3, g/e = -1, match or mismatch - from PAM250 substitution matrix below.  SW algorithm.    1. Complete the scoring matrix.    Scoring matrix with PAM250 scores:   R I V L F         M         K           2. Set up, initialize and complete the SW matrix.  3. Retrace, align and score alignment(s).             Use the arrows and circles for the matrix and path(s).                                                                                                                                                                                                                                                                                                                                 R I V L             F           M           K           Align and score all optimal alignments here.  PLZ  the arrows and circles for the matrix and path(s) AND SHOW ALL possible Alignment  Here the following points…

Task 1. Your graduate advisor asks you to amplify the following sequence of DNA by PCR: 5’-ATACGCATTCGGACCAGGTCCTAA-3’ 3’-TATGCGTAAGCCTGGTCCAGGATT-5’ a. To ensure that the entire sequence above is amplified, what 6-nucleotide DNA primers should you add to your PCR mix?   You order the primers listed above, but instead receive the following set of primers: 5’-CGCATT-3’ 5’-GGACCT-3’ b. What portion of the double stranded DNA molecule will be amplified after 10 rounds of PCR?   Your labmate attempts to rescue your PCR reaction by providing you with the following set of primers: 5’-ATACGC-3’ 5’-TCCTAA-3’ c. What is the result of running the PCR reaction with your labmate’s primers? How many double stranded molecules of DNA will result from 10 rounds of amplification?

Search (Option + Q) Review View Help O Editing v A BIU D Av A ... Question 4 The use of physical or chemical nonviral transfer of genetic material is called Transfection Transduction Transformation Trangenesis Question 5 Congenital hearing loss is mostly autosomal recessive. When two persons of congenital hearing loss marry, as they often do, their biological children have normal hearing. The reason for the children to have normal hearing is that one of the parents is homozygous recessive for one gene and the other parent is homozygous recessive for another gene all the normal children have reverse mutations in their DNA each of the parents has reverse mutations in the germline cells early in their life. all normal children are homozygous recessive. Question 6 The enzyme responsible for transcribing complementary DNA from MRNA is DNA Polymerase Reverse transcriptase RNA Polymerase Endonuclease

acconline.austincc.edu/ultra/courses/_891351_1/cl/outline O gel electrophoresis O PCR O genomic library DNA microarray A patient is suspected of having a certain type of cancer that involves several genes. Doctors want to determine whether this patient is expressing these cancer genes and to what leve each gene is being expressed. What technique may be used? O molecular cloning O CRISPR gene editing 5 genetic recombination that occurs naturally (think horizontal gene transfer, meioșis, fertilization, or mutations). Ar : Y EV АУ ΩΘΙ 用く X A MacBook Air & 8 88 Q V Ix % 0 8A EXE D B 图 Q 用图 5 d Show All † 19 EHR Immuniza

1. Definitions and brief statements 1. DNA Data Bank of Japan 2. MeSH Database 3. Please describe the 3 steps and temperature that are cycled during a PCR reaction. 4. What are global alignment and local alignment and their algorithms? ( 5. ProtScale

Restriction mapping of the delta chromosome I need help with question two please

patibility Mode] UE Layout References Mailings Review View A A Aa vA 前、一间刷 一| T AaBbCcDdE AaBE x A Av X, Normal No Spacing Headi co Edit and Save Start your free one month trial of Microsoft 365 or sign in to activate an existing subscription. 9. In human beings, the gene for red-green colorblindness (r) is sex-linked and recessive to its allele for normal vision (R), while the gene for freckles (F) is autosomal and dominant over its allele for nonfreckled (f). A nonfreckled, normal-visioned woman whose father was freckled and colorblind, marries a freckled, colorblind man whose mother was nonfreckled. a. What is the genotype of the woman's father? b. What is the probability that the couple's first child will be a non-freckled, normal visioned girl c. What is the probability that the first two children born to the couple will be freckled and colorblind girls? d. What is the probability that the first child born to the couple will either be a freckled, colorblind boy or a non-freckled,…

Please answer fast Using the cDNA copies of the transcripts from parasite-cells infected with red fluorescence, and cDNA copies of the transcripts from normal cells labeled with with green fluorescence, what color would you predict the well with the ninjurin gene would appear on the microarray?   a-gray b-green c-red d-yellow

Question:- 2. How could multi-genes families complicate things in terms of using CRISPR to knock out a target gene and achieve a target phenotype?

Complete the following problems. Restriction enzymes (REs), which cut D NA at specific sequences, are classic tools in molecular biology. Because of their specificity in cutting DNA, REs can be used to "map" DNA sequences by analyzing the fragments generated upon restriction digest, as in the example shown in Figure 1. Your task is to study the circular plasmid, pMBBS, through restriction digests. You subjected the pMBBS plasmid to complete digestion by different combinations of three REs (EcoRI, BamHI, and Xhol), and analyzed the results on an agarose gel, shown below. Using the data you can glean from this gel, answer the questions that follow. EcoRI BamHI Xhol *The same total amount of DNA was loaded in each lane. 1. What is the total size of the pMBBS plasmid in bp? Answer: bp + DNA size ladder 2. How many cut sites on the PMBBS plasmid does each RE have? EcoRI: BamHI: Xhol: 3000 bp 2500 bp -2000 bp -1500 bp 1200 bp 1000 bp 900 bp 800 bp 700 bp 600 bp 500 bp 400 bp 300 bp 200 bp…

STEP BY STEP Exaplanation would be appreciated so I can understand for upcomming exam. In humans, the COL1A1 locus codes for a collagen protein found in bone.  A recessive COLA1 allele believed to reduce bone density and increase risk of fractures differs from the wild type allele by the presence a GT in the first intron of the gene. This mutation can be easily screened by PCR, using COL1A1-specific primers followed restriction enzyme digest of the product with MscI. The normal allele is denoted as G and the recessive allele as T.  A recent study of 894 women found that 570 were GG, 291 were GT and 33 were TT.  (Assume for this exercise that these values reflect both male and female values for the sampled population. What are the frequencies of each allele? What are the frequencies of each genotype? c. Is this locus in Hardy-Weinberg equilibrium? Use the table below to evaluate the significance of any deviation from H-W expectations.

Instructions: Read 13-2 Manipulating DNA pages 322-323. As you read each section, examine the figures and captions (explanations). Identify any questions you may have. 1) Develop an analogy for the processes researchers use to make changes to DNA. In yo analogy, explain how it is similar to the techniques used in genetic engineering. You can draw a graphic organizer, make a table, or write a few sentences describing your analogy. 2) Devise flowchart that shows the steps to prepare DNA for gel electrophoresis, as well the protocol for setting up and running a gel. You can add diagrams to the flowchart an add detailed notes if you like. English (inited Sate) O Focs ere to search 4 CO RU G\ L B. 2N A\ Alt Ciri

Part A. If student counts 63 colonies on their 10^-6 dilution LB plate. What was the original concentration of their cells if they plated 100ul? Part B.If we used pGFPuv as the template for PCR positive control. This is because: a. it contains the GFP gene so it should show a product. b. It contains DNA fragments that were added to the ligation reaction. c. It is the desired plasmid we wanted to make. d. So we have a band to compare our unknown plasid to allowing us to check if the unknown is the right size.

Select all that would be true if I had a missense mutation in an gene: The missense mutant protein would be the same size by Western as the wildtype protein The missense mutant allele would be a different size compared to wildtype by PCR- electrophoresis The missense mutant protein would be a different size by Western compared to the wildtype protein The missense mutant allele would be the same size as wildtype by PCR-electrophoresis

QUESTION: There is an RFLP pattern that belongs to a disease. I need to find an inheritance pattern and marker related to the disease..

1. Definitions and brief statements 1. DNA Data Bank of Japan 2. MeSH Database 3. Please describe the 3 steps and temperature that are cycled during a PCR reaction. ✓ V V 4. What are global alignment and local alignment and their algorithms? ( 5. ProtScale

I need help with a biology lab question. What are the four componets required for PCR to work?     thanks

Answer each of the following correctly. Designer Genes Work (This is all about Applications of Recombinant DNA). 1. How does DNA Replicate?(1-3 sentences only) 2. What is Genetically Modified Organism (GMO)?(1-4 sentences only) 3. Illustrate your own Designer genes using this information:The Arctic apple is a fruit engineered to resist browning after being cut. Currently they are only available in the US – in golden, fuji and gala varieties – where they have been given Food and Drug Administration approval. If approved in Europe, they would have to be labelled as genetically modified. The manufacturers claim the main benefit is to help cut down on food waste. And based on the following: 1. Identify a special trait. 2. Identify a source organism. 3. Identify a target organism 4. Identify the modified/added trait. Example: Hot Tomato > Chili > Tomato > Spicy Tomato It was reported this week that Brazilian scientists are hoping to create spicy tomatoes using Crispr…