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[answered] 1. You want to make an agarose gel to run a DNA digestion t


Microbiology stock concentration questions, please provide all calculations. THANKS


1. You want to make an agarose gel to run a DNA digestion that you have prepared, but you do not have the

 

reagents needed to make it. First you must prepare a variety of stock solutions that are needed to make 1X TAE.

 

How would you accomplish the following?

 

a. Prepare 600 mL of a 40x Tris acetate (MW = 121.14) solution that will have a final concentration of 1mM. b. Prepare 500mL of a 40x EDTA solution (MW = 372.24) that will have a working solution of 2mM. c. Now you want to prepare 1L of 20x TAE using your stock solutions from a) and b). What would you do? d. Now that you?ve made up your 20x stock solution of TAE using the reagents you just made in questions a. and

 

b., how much of your 20x TAE stock solution and water would you use to make 500mL of 1x TAE? e. Using your 1x TAE you make two agarose gels, each 40mL. How much of the 1x TAE and agarose would you

 

need to make a 0.7% gel and a 1.5% gel? f. You dissolve your agarose into the TAE and allow the mixtures to cool. Calculate how much ethidium bromide

 

you need to add to the gels. The rate you need to add EtBr is 2?L per 100mL of agarose gel mixture.

 

How many ?L did you add to your gel? If your EtBr stock is 10mg/mL, how much (in ?g) did you add to your 40mL mixtures? 2. a. Make 500mL of NaOH (MW = 39.99) that will have a final concentration of 0.1M. b. What is the maximum amount of 25mM NaOH solution that can be made using all 500mL of 0.1M stock? 3. What is the percentage of NaCl (MW = 58.44) in a 500mL solution with 20g of NaCl in it? 4. How much glucose and water is added to make 500mL of a 15% glucose solution? (MW = 180.16) 5. Make 1L of a 5x NaCl solution (MW = 58.44) that will have a final concentration of 1x. 6. You plan to prepare Luria Broth so that you can make LB and LA plates. You make 500mL of LB, and divide it

 

into two containers ? half for the LB and the other half for LA plates. How much agar will you need to add to the

 

container for plates so that they will be 1.5% agar? You then use the LB and plates to do a serial dilution experiment as well as a plaque assay.

 

a. For the serial dilution experiment you take 10?L of an E. coli culture and dilute it in 990?L LB. You do a total of

 

4 dilutions and plate 100?L of each dilution on a separate LA plate. You see 29 colonies on the second to last

 

plate. What is the cell concentration of the original bacterial culture? b. How many colonies would you see on the last plate? c. For the plaque assay, your T4 bacteriophage titer is 7.8x10 8 pfu/mL. You do six 1:10 dilutions of the virus,

 

putting 10?L of each dilution in a tube of top agar with 100?L of E. coli. After pouring the contents of the top

 

agar onto an LA plate, you incubate overnight. How many plaques would you expect to see on the final plate? 7. You are making a PCR reaction mixture with a total volume of 50L. The primer set concentration is 1?l

 

primer/10?l reaction volume. The plasmid stock solution concentration = 0.01?g/?l, of which you need 100ng.

 

How much of each PCR reaction component will you add, given the following?

 

5x Mastermix __________

 

Primer set __________

 

DNA template __________

 

H20 _____________

 

8. If you start out with 25 DNA fragments of your target gene in your pcr reaction, how many fragments will you

 

have after 30 cycles?

 


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