4. Create a PCR master mix for the following: 48 samples, 25 uL total volume per reaction, 2 uL templateDNA volume, 2.5mM MgCl, 0.5 µM each primer. Your stock concentrations are: reagent mix (2X, doesnot contain MgCl,), primers (F/R; 10 uM), MgCl, (50 mM). The master mix calculation must include a10% overage, and all answers are to be given to one decimal place.

PCR, or the polymerase chain reaction, is a chemical reaction that is used by molecular biologists…

Answered: 4. Create a PCR master mix for the…

Biochemistry

9th Edition

ISBN:9781319114671

Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Chapter1: Biochemistry: An Evolving Science

Section: Chapter Questions

Problem 1P

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1. The final amount of each primer required in a PCR reaction is 0.025 nanomol. If the total volume of the PCR reaction is equal to 100 µl and the stock concentration of each primer is equal to 10 µM. Calculate the volume of stock primer that needs to be added in order to ensure a primer amount of 0.025 nanomol. 2. A 250 ml phosphate buffer solution contains 204.12 µg KH2PO4 and 87.09 µg K2HPO4, respectively. Determine the molarities in micromolar (µM) of the abovementioned compounds in the phosphate buffer solution. [Mw KH2PO4 = 136.08 g/mol and Mw K2HPO4 = 174.18 g/mol] 3. You are required to use the pET 28b expression vector in a cloning experiment. If the vector is 5.368 kilobasepairs in length and the mass of the DNA that you have added represent 8.5 x 109 molecules. Determine the mass of vector DNA that you have added to your reaction? [1basepair ~ 660 Da or g/mol, Avogadro’s constant = 6.023 x 1023 molecules/mol]
Complete this Master Mix table for 8 DNA samples, a positive control, negative control, and an extra reaction for pipetting error. Show your work. Master Mix Conc. Of Total μL µL/Rxn (total per tube) Final Number of Stock Conc. Reactions needed for solution master mix PCR buffer 50X 1X Water DNTP mix 100 mM 200 µM MgCl2 Forward 24 mM 2.5 mM 2 μΜ 0.1 μΜ primer Reverse 2 μΜ 0.1 μΜ primer Taq polymerase DNA 5 U/uL 0.03 U/ µL 1 μL 60 μL Total volume of the entire reaction (µL)
5' 3' O 60°C 5' O 95°C Primer 1 O 75°C ORF For your PCR reaction above, the primer set has a much higher AT (low GC) content than what would be considered normal. Due to this difference in AT content, the optimal annealing temperature for the primers is going to have to be altered from the "normal" temperature cycle: 95°C 60°C ⇒ 75°C Which of the temperatures will have to be altered to accommodate this difference the primer set? Primer 25¹ 3' 5' annealing with
1. You decide to perform dideoxy sequencing on a PCR product. You add the appropriate 32P- labeled primer, DNA polymerase, DNA template (the PCR product), buffer, DNTP mix, and a small amount of one of the four ddNTPs to four reaction tubes. You run the reactions in the thermal cycler, load each reaction into a separate lane of a polyacrylamide gel, and separate the products by gel electrophoresis. In the figure below, the lanes are labeled according to the ddNTP added. ddATP ddTTP ddCTP ddGTP - Lane 1 2 3 4 a) In lane 5, draw what you would expect to see if you prepared a reaction using a nucleotide mix containing only DATP, DTTP, dCIP, and dGTP. b) In lane 6, draw what you would expect to see if you prepared a reaction using a nucleotide mix containing only ddATP, DTTP, dCIP, dGTP. ||
Below are several problems frequently faced by researchers when running the PCR. Give one (1) solution to each problem stated. 1. No PCR product 2. Multiple bands appeared after gel electrophoresis (you are amplifying just 1 gene) 3. Bright thick bands at the end of the agarose gel after electrophoresis 4. Bands appeared at the negative control
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
4 of 11 Calculate the annealing temperature (Tm in °C) for the PCR primer below. Do not include units when submitting your answer. 5'-ACT CAA AAC ACT AGT TGC-3' Answer:
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
Match the PCR sample with the predicted result on the agarose gel. 1) Sample from individual homozygous for their PV92 locus with the Alu insert. Five bands of 100, 200 500, 700 and 1000bp Single band of 300 bp Two bands one of 300 bp and one of 641bp Tow bands one of 641 bp and one of 941 bp No Bends Single band of 941 bp Single band of 641bp 2) Sample from individual homozygous for their PV92 locus without the Alu insert. Five bands of 100, 200 500, 700 and 1000bp Single band of 300 bp Two bands one of 300 bp and one of 641bp Tow bands one of 641 bp and one of 941 bp No Bends Single band of 941 bp Single band of 641bp 3) Sample from individual heterozygous for their PV92 locus having the Alu insert. Five bands of 100, 200 500, 700 and 1000bp Single band of 300 bp Two bands one of 300 bp and one of 641bp Tow bands one of 641 bp and one of 941 bp No Bends Single band of 941 bp Single band of 641bp 4) Negative control sample. Five bands of 100, 200 500, 700 and 1000bp…
1. Primers come at a concentration of 200 pmol//ul (uM). You want the final concentration of your primers to be 0.2 uM. How would you dilute the primers in water so that you can add 1 ul in a PCR mix with a total volume of 50ul?
1. Explain what primers are and what purpose they serve in a PCR reaction. Explain the main steps and temperatures of one PCR cycle. 2. Explain the purposes of the following components of gel electrophoresis: agarose, loading buffer, DNA ladder/marker, electrode buffer, electrodes. 3. Explain the expected results of the Alu sequence at PV92 that we tested, in terms of numbers of bands and their sizes. A labeled drawing would suffice.
A student is trying to add 9.0 pmol of primer mix to a 20.0 µL PCR. The primer mix is at a concentration of 50.0 µM, and the student determines that a serial dilution is required because directly adding the primer mix would require a volume less than 1.00 µL. The student takes 1.40 µL of the primer mix stock and mixes it with 25.0 µL of ultrapure H2O. Calculate the volume (in µL) of the intermediate solution required to add the correct amount of primer mix to the PCR.

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