QUESTIONS FOR PCR LAB
1. How many molecules of a 3 kb plasmid are in 1 ng of sample?
2. Given:
Stock solutions:
Reaction Buffer = 10X
DNA concentration = 20 µg / ml
dNTP concentration = 10 mM
Taq pol = 0.5 Units/ µl
Final reaction volume = 100 µl
Final amount of DNA template= 100 ng
Final dNTP concentration= 200 µM
Final amount of Taq pol = 2 units
Please fill in the volumes in the table below.
10 X Buffer
DNA
dNTP
Taq
Water
3. Rank the following primers with regard to which will allow a higher annealing temperature in
a PCR reaction. Assume all have perfect complementarity with their targets. Rank 1(highest
annealing T) to 3 (lowest annealing T).Explain your ranking.
Primer X—AGTCCGTCGTAACGGAG
Primer Y--- AGTCAATCGTAACGAAG
Primer Z--- AGTCCGTCGTAACGGAGAATTAGCTATGCA
4. A researcher is doing a PCR-based experiment using the following temperature profile:
 Melt for 1 minute at 94C
 Anneal for 30 seconds at 37C
 Extend for 30 seconds at 70C
Unfortunately, her gels show way too many bands. Her primers have perfect complementarity to
the target sequence. Give a simple way to reduce the number of bands.
5. Calculate the amount of ethidium bromide (5 mg/ml) you need to add to a 200 ml, 1.5% TAE
agarose gel to achieve a final [EtBr]= 100µg/ml in the gel. Show all work.
6. The lab procedure originally called for you to dilute your sample in you to dilute your sample
until you had 1 femtogram/µl (1X10-15 g/µl) of sample. Assuming you start with a 5 µg/ µl
sample, describe the steps you would take to get 4 samples of 100 ng/µ1, 1 ng/µ1, 10
picogram/µl (1X10-12 g/µl), and 100 femtogram/µl (1X10-15 g/µl)) final concentration.