Question

In: Biology

7.You want to set up a PCR reaction. The protocol suggests a 50 µL reaction volume...

7.You want to set up a PCR reaction. The protocol suggests a 50 µL reaction volume containing 1X buffer, 200 µM dNTPs, 0.5 µM each primer (forward and reverse), 250 ng template DNA, and 1 U enzyme.

You have the following reagents: 5 X buffer, 10 mM dNTPs, 10 µM each primer (forward and reverse), 125 ng/µL template DNA, and 2 U/µL enzyme.

How many µL of water, reaction buffer, dNTPs, each primer, template DNA and enzyme would you combine to set up that reaction? (note that the order I’ve listed these components is the order you’d add them to the reaction)

Expert Solution

Ans. #I. Buffer:

Using C1V1 (5X stock buffer) = C2V2 (final PCR mix, 1X, 50 uL)

Or, 5X x V1 = 1X x 50.0 uL

Or, V1 = (1X x 50.0 uL) / 5X

Hence, V1 = 10.0 uL

Hence, required volume of 5X stock buffer = 10.0 uL

#II. dNTPs

Using C1V1 (10 mM stock dNTPs) = C2V2 (final PCR mix, 200 uM, 50 uL)

Or, 10 mM x V1 = 200 uM x 50.0 uL ; [1 mM = 1000 uM]

Or, 10000 uM x V1 = 200 uM x 50.0 uL

Or, V1 = (200 uM x 50.0 uL) / 10000 uM

Hence, V1 = 1.0 uL

Therefore, required volume of 10 mM stock dNTPs solution = 1.0 uL

#III. Primers

Using C1V1 (10 uM stock dNTPs) = C2V2 (final PCR mix, 0.5 uM, 50 uL)

Or, 10 uM x V1 = 0.5 uM x 50.0 uL

Or, V1 = (0.5 uM x 50.0 uL) / 10.0 uM

Hence, V1 = 2.5 uL

Therefore, required volume of 10 uM stock primer solution = 2.5 uL

#IV. Required volume of stock template DNA =

Required amount of template DNA/ Concertation of stock template soln.

= 250.0 ng / (125.0 ng/ uL)

= 2.0 uL

#V. Required volume of stock enzyme solution=

Required amount of enzyme/ Concertation of stock enzyme soln.

= 1.0 U / (2.0 ng/ uL)

= 0.5 uL

#VI. Required volume of water = 50.0 uL – (summed vol. of all other ingredients I-V)

= 50.0 uL – (10.0 uL + 1.0 uL + 2.5 uL + 2.0 uL + 0.5 uL)

= 50.0 uL – 16.0 uL

= 34.0 uL

gladiator answered 1 year ago

You want a 50 µL reaction volume containing 1X reaction buffer, 1 µg DNA, and 10...

You want a 50 µL reaction volume containing 1X reaction buffer, 1 µg DNA, and 10 Units of enzyme.You have a plasmid with a concentration of 250 ng/µL, 10X reaction buffer, and your enzyme concentration is 20,000 units/mL. How many µL of water, reaction buffer, DNA and enzyme would you combine to set up that reaction? Please show work

Question 1: You set up a 200 µL PCR to amplify a 1-kb fragment by adding...

Question 1: You set up a 200 µL PCR to amplify a 1-kb fragment by adding 20 µL of a 500 nM primer that is 20-bp in length. How many times would you expect to find the exact 20-bp sequence in the human genome (using a genome size of 3.0 E+9 bp) at random? How many moles of the primer have you added? How many molecules of that primer have you added? Describe whether this amount of primer is a...

You now set up a ligation reaction with the following components: • Plasmid Vector (0.05 µg/µL)...

You now set up a ligation reaction with the following components: • Plasmid Vector (0.05 µg/µL) • Insert DNA (0.2 µg/µL) • 10X Ligase Buffer • Water Because you are unsure what the best ratio of insert to plasmid is, as this is a new experiment for you, you plan to set up two experiments, one with a 3:1 ratio of insert to plasmid, and one with a 6:1 ratio of insert to plasmid. In both cases the amount of...

You are setting up a PCR reaction using DNA from a bacteria with a genome size...

You are setting up a PCR reaction using DNA from a bacteria with a genome size of 17 megabase-pairs (mb). How many nanograms of DNA will you need to add to the reaction to ensure there are at least 100 copies of the single gene you are trying to amplify? (assume the average molecular weight of a nucleotide pair is 650). (2 pt)

Instead of following the incubation directions listed in your lab protocol, imagine that you set up...

Instead of following the incubation directions listed in your lab protocol, imagine that you set up your plates and then left them incubating for over a week before you looked again at them. What problems could this cause for your MSA plates? How about for your EMB and MacConkey plates?

You want to set-up a restriction enzyme digestion of a plasmidDNA sample. You would like...

You want to set-up a restriction enzyme digestion of a plasmid DNA sample. You would like to digest 0.75ug of this DNA with 50 units of BamHI and 50 units of HindIII in a total volume of 25uL containing 1x buffer A.How much (in uL) of the following stock solutions and water are needed?a) 200ng/uL plasmid DNAb) 10x buffer Ac) 50,000 units/mL BamHId) 25,000 units/mL HindIIIe) water

Suppose the DJIA stands at 24, 200. You want to set up a long straddle by...

Suppose the DJIA stands at 24, 200. You want to set up a long straddle by purchasing 100 calls and an equal number of puts on the index, both of which expire in three months and have a strike of 242. The put price is listed at $4.20 and the call sells for $5.20. a. What will it cost you to set up the straddle, and how much profit (or loss) do you stand to make if the market falls...

1. If you started a PCR protocol with 10 double-stranded molecules of DNA, how many double-stranded...

1. If you started a PCR protocol with 10 double-stranded molecules of DNA, how many double-stranded molecules of the product would you have after 5 cycles of PCR? 2. What is the purpose of using DNA polymerase from Thermus aquaticus for the polymerase chain reaction rather than a DNA polymerase from a better characterized bacterium such as E. coli?

Set up (Do Not Evaluate) a triple integral that yields the volume of the solid that...

Set up (Do Not Evaluate) a triple integral that yields the volume of the solid that is below the sphere x^2+y^2+z^2=8 and above the cone z^2=1/3(x^2+y^2) Rectangular coordinates b) Cylindrical coordinates c) Spherical coordinates

Set up an integral that uses the disk method to find the volume of the solid...

Set up an integral that uses the disk method to find the volume of the solid of revolution obtained by revolving the area between the curves y = sech(x/2), y =2, x =0 and x = 4 around the line y=2. Include a sketch of the region and show all work to integrate and. Note: Recall that sech(u) = 1/cosh(u). Please show details for every single step