. If you forgot to add dNTPs to a sequencing reaction, what would be the result? Only very long fragments would be synthesized Only very short fragments would be synthesized The fragments would not be labelled DNA polymerase would be inactivated Sequencing would proceed normally
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- Which of the following best describes the process of DNA sequencing? a. DNA is separated on a gel, and the different bands are labeled with fluorescent nucleotides and scanned with a laser. b. A laser is used to fluorescently label the nucleotides present within the DNA, the DNA is run on a gel, and then the DNA is broken into fragments. c. Nucleotides are scanned with a laser and incorporated into the DNA that has been separated on a gel, and then the DNA is amplified with PCR. d. Fragments of DNA are produced in a reaction that labels them with any of four different fluorescent dyes, and the fragments then are run on a gel and scanned with a laser. e. DNA is broken down into its constituent nucleotides, and the nucleotides are then run on a gel and purified with a laser.Look at each PCR component listed below. For each one, determine which steps(s) of the PCR reaction (denaturation, annealing or extension) would be directly affect if that component were missing. Taq polymerase: Oligonucleotide primers: DNA template: Deoxynucleotides (A, T, G and C): Imagine that you correctly prepare your PCR reaction mixture, but there is something wrong with the thermal cycler. Describe what would happen if: The thermal cycler was stuck on 940C: The thermal cycler cycled between 600C and 720C, but never reached 940C The thermal cycler cycled between 940C and 720C, but never reached 600Put the steps of one PCR cycle in the correct order: The PCR reaction mixture is heated to about 70 degrees, which is the optimum temperature for the polymerase to build the new strands of DNA, starting at the 3' end of the primer. The PCR reaction mixture is heated to 95 degrees Celsius, which denatures the double stranded template DNA. The PCR reaction mixture is cooled to about 50-55 degrees, which allows the primers to find their complementary site on the template and "anneal" the
- Imagine that you are producing a PCR product of approximately 2.1 kb in size with constant non-specific products which are two very light bands present above the desired product and another intense band below the desired product. Now for an experiment, you need very pure products out of the PCR process. To avoid non-specific product production, you want to take a different PCR approach. There are two PCR processes that your supervisor has recommended you which are: Touchdown PCR technique and Gradient PCR technique. Considering that you need the pure product only once for your future experiment, mention in no more than 5 sentences which PCR technique will you choose and why?Which of the following is necessary for a PCR reaction to proceed? a) the sequence of the ends of the DNA to be amplified must be known. b) the sequence of restriction endonuclease recognition sites in the DNA to be amplified and in the plasmid, where the amplified DNA fragment will be cloned must be known. c) The complete sequence of the DNA to be amplified must be known. d) The sequence of restriction endonuclease recognition sites in the DNA to be amplified must be knownA student carries out PCR using the following steps: step 1: 94C for 1 minuteStep 2: 60C for 30seconds Step 3: 72C for 30 seconds The student is examining a 1000-bp DNA fragment as part of their research project. If the limit of detection of this molecule 9 x 108 molecules, what is the minimum number of PCR cycles you would have to run to detect PCR product generated from a single molecule of the template? 2. Two double-stranded fragments of DNA are exactly the same length. At 89 C, fragment A has completely denatured which means the two strands have separated. At that temperature, fragment B is still double-stranded. How might these fragments differ to result in different denaturation temperatures?
- You wish to amplify a segment of DNA from a plasmid template by PCR with the use of the following primers: 5’-GGATCGATGCTCGCGA3' and 5' -AGGATCGGGTCGCGAG-3'. Despite repeated attempts, you fail to observe a PCR product of the expected length after electrophoresis on an agarose gel. Instead, you observe a bright smear on the gel with an approximate length of 25 to 30 base pairs. Explain these results.A student is trying to add 15.0 ng of DNA template to a 20.0 µL PCR. The DNA template is at a concentration of 65.0 ng µL-1, and the student determines that a serial dilution is required because directly adding the DNA template would require a volume less than 1.00 µL. The student wants to prepare an intermediate solution at a concentration of 15.0 ng µL-1. If the DNA template stock will be mixed with 13.0 µL of ultrapure H2O, calculate the volume (in µL) of the DNA template required to prepare the intermediate solution.Ben used the following primer pairs to amplify the COI region of his DNA template: Afterwards, he used Agarose Gel Electrophoresis to visualize the PCR products. Based on the gel: a) Was he able to successfully amplify the desired region in all replicates? b) Are there errors in Ben's gel? Does he need to repeat his PCR?
- The answer is option A In the PCR amplification, two primers are used to flank the target region to amplification. The two primers are forward primer and reverse primer. The forward primer binds to the template DNA and the reverse primer binds to the complementary strand. Step 2 In the above question, the two sequences of 21bp primers in the 5' to 3' Orientation that allows amplifying the given gene sequence are : ATGTTTGTTTTTCTTGTTTTA and GTCAAATTACATTACACATAA CAN YOU FURTHER EXPLAIN WHY IT IS "GTCAAATTACATTACACATAA," I don't understand why it's called reverse primer? and where exactly it is binding to on the strand? (A picture would really help)Why is it important to sequence positive clones derived from PCR cloning? Group of answer choices Errors could have been incorporated during restriction enzyme digestion and so it is important to verify that only the expected protein is produced. Errors could have been incorporated during plasmid DNA extraction and so it is important to verify that only the expected protein is produced. Errors could have been incorporated during PCR and so it is important to verify that only the expected protein is produced. Errors could have been incorporated during cloning and so it is important to verify that only the expected protein is produced..How much PCR product (500 bp) should be added to a ligation in which 50 ng of Vector (6.6 kb) vector will be used if a 3:1 insert:vector molar ratio is desired? Vector concentration is 50 ng/uL and insert concentration is 35 ng/ul. Provide mass and volumes for the ligation reaction