Question

PCR Optimization (lab write up due next week) In our lab experiment you will do a PCR / PCR optimization experiment. The key components of a PCR reaction include a DNA Template that is to be copied, Forward and Reverse Primers at many fold higher concentration, an equal mixture of each of the 4 dNTPs, Taq Polymerase and a buffer to maintain the optimal pH for Taq Polymerase. You will be amplifying a 2kb beta-galactosidase gene from p?NH/bgaB. Note where the Forward and Reverse Primers bind to the plasmid (position 1-22 and 2369-2345. This yields a 2323 bp product. pDaNH-BgaB 4457 bp Directed Evolution Forward Primer Binding Site (Tm 73.7) 1-22 Directed Evolution Reverse Primer Binding Site (Tm 70.7) 2369-2345 The primers that you will be using are: Forward: 5’-TTTCCAGAGGCCGATTAGGTACAC-3’ Reverse: 5’-GTCGTACCGATAGCTCGAAACGAC-3’ As is common for most DNA binding enzymes, Mg2+ ions are found to stabilize the interaction between the polymerase and DNA and thus acts as a cofactor. The exact concentration pf Mg2+ that is optimal for a PCR reaction varies and the purpose of this lab is to determine the optimal concentration of MgCl2 to use in our future PCR reactions. 1. From the attached materials, determine the Tm for each primer. 2. From that, determine the appropriate annealing temperature. 3. Given the distance between the Forward and Reverse primer sites on the plasmid is 2368 bp Use this number, determine the extension time required at 72 C. 4. Use the answers above to complete table 2.

PCR Optimization (lab write up due next week)
In our lab experiment you will do a PCR / PCR optimization experiment. The key components of a PCR reaction include a DNA Template that is to be copied, Forward and Reverse Primers at many fold higher concentration, an equal mixture of each of the 4 dNTPs, Taq Polymerase and a buffer to maintain the optimal pH for Taq Polymerase.
You will be amplifying a 2kb beta-galactosidase gene from p?NH/bgaB. Note where the Forward and Reverse Primers bind to the plasmid (position 1-22 and 2369-2345. This yields a 2323 bp product.
pDaNH-BgaB 4457 bp
Directed Evolution Forward Primer Binding Site (Tm 73.7) 1-22
Directed Evolution Reverse Primer Binding Site (Tm 70.7) 2369-2345
The primers that you will be using are:
Forward: 5’-TTTCCAGAGGCCGATTAGGTACAC-3’
Reverse: 5’-GTCGTACCGATAGCTCGAAACGAC-3’
As is common for most DNA binding enzymes, Mg2+ ions are found to stabilize the interaction between the polymerase and DNA and thus acts as a cofactor. The exact concentration pf Mg2+ that is optimal for a PCR reaction varies and the purpose of this lab is to determine the optimal concentration of MgCl2 to use in our future PCR reactions.
1. From the attached materials, determine the Tm for each primer.
2. From that, determine the appropriate annealing temperature.
3. Given the distance between the Forward and Reverse primer sites on the plasmid is 2368 bp Use this number, determine the extension time required at 72 C.
4. Use the answers above to complete table 2.

Added by Victor B.

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