Question

Just need 3-6. NOT 1-2

A research associate working for Intragene Therapeutics prepares an investigative PCR reaction to screen human tissue samples for specific mutations. He uses 55 ng of genomic DNA from one tissue sample in a single PCR reaction, to amplify a 1,246 bp fragment. The PCR protocol requires a final concentration for two primers of 1.5 uM each, and a final concentration of 180 uM for dNTPs, in a total PCR reaction volume of 65 uL. During the PCR process, the fragment is amplified for 33 cycles. He quantifies the amount of PCR products to be 74 ng/uL.

1. If his purified genomic DNA template stock has a concentration of 1.53 mg/mL, and he dilutes it 1:100, how many microliters of this dilution does he use? Answer to one decimal place.

2. He decides to dilute his DNA template stock so that he can pipet a 15 uL volume that contains 55 ng of genomic DNA into his PCR reaction. If he starts with 1 uL of the DNA template stock, what is the total volume of the dilution he needs to make? Answer to the nearest whole number.

3. How many copies of the fragment are produced in his PCR reaction? Assume that the target sequence is present in only one copy in the genome, as in a haploid human genome. Answer to the nearest whole number.

4. What is the amount of amplification of the PCR product in his reaction ‐ that is, the fold increase in the amount of amplified DNA segment? Answer in decimal notation.

5. If one of the primer stocks in his lab has a concentration of 50 uM, what volume (in microliters) does he add of this primer to have the final concentration specified in the PCR protocol? Answer to the nearest whole number.

6. The dNTP master mix available in the company lab has a concentration of 12 mM total dNTP. What volume, in microliters, of this master mix does he add to his PCR reaction to have the final concentration specified in the PCR protocol? Answer to the nearest whole number.

Answer 1