Question

D. After you run your PCR reaction, what next steps would you perform to determine that the PCR amplified the region of interest? E. After your procedures in step D above, you decide to measure the concentration of DNA in your PCR product. Briefly explain how you would do this given the tools we have available. What values would you expect to confirm that you have a good DNA concentration and purity? F. Write and briefly describe a program you would use to amplify your sequence of interest if the expected size is 1 kb, the primers have a Tm of 62 °C, and the polymerase you are using extends at 68°C. (There is not one exactly right answer for this, but some answers may be better than others.) 3. Your instructor asks you to refill your spray bottle of 70% EtOH which holds 425 ml. You only have 95% EtOH available in the lab. How much 95% EtOH do you need? How much dH20? Show your calculations. C1V1 = C2V2

Answer 1

D) after the completion of PCR to determine the amplification of desired product we will run the gel. In gel we will load DNA ladder along with PCR product. And after run , size of the amplified fragment is determined using DNA ladder.

E) after observing the desired DNA fragments. We will run gel , and try to load whole PCR product. After run of gel, PCR product is eluted from the gel. And DNA is eluted from the gel using appropriate methods and dissolved in pure water. Concentration of DNA and purity will be determined using Nanodrop. Nano drop concentration=  1.8 denotes pure DNA, values higher side denotes RNA contamination while lower side values show protein contamination.

F) PCR protocol

Step 1 94° c - 2.0 minute

Step 2 94° C --- 15 second denaturation

62 °C - 15 second annealing

68°C -- 1 minute extension

Step 2 repeated 30- 35 cycle

Step 3 72°C - 2 minute ( stop of reaction)

4°C  

3) C1V1 = C2V2

C1= 95% EtoH C2= 70 % EtoH

V2 = 425 ml.

95Xa = 70X 425ml

A= (70X 425)/ 95 = 313ml

Add 313 ml 95% EtoH and make to 425 ml with water

313ml 95% EtoH + 112ml water