LAB 4 WRITE-UP
- Lab coat and disposable gloves
- PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s(http://www.promega.com/resources/protocols/product-information-sheets/g/gotaq-colorless-master-mix-m714-protocol/)
- DNA/ primer mix, 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer
- A strip of empty PCR tubes
- Disposable pipette tips: only use each only once. Never re-use disposable pipette tips or samples will be cross-contaminated
- Cup for discarded tips
- OpenPCR machine: shared by two groups
PCR Reaction Sample List
OpenPCR Program Settings
The following values were used to run a heating & cooling program on the thermal cycler.
- HEATED LID: 100°C
- INITIAL STEP: 95°C for 2 minutes
- NUMBER OF CYCLES: 35
- Denature at 95°C for 30 seconds
- Anneal at 57°C for 30 seconds
- Extend at 72°C for 30 seconds
- FINAL STEP: 72°C for 2 minutes
- FINAL HOLD: 4°C
Research and Development
The Function of Each Component of a PCR reaction-
- Template DNA- This provides a set of nucleotides for the primers to bond to and then to be replicated.
- Primers-These sets of nucleotides bind to the template DNA and begin the replication process.
- Taq Polymerase- This enzyme combines nucleotides to make a new strand of DNA which replicates the template strand
- Deoxyribonucleotides (dNTP's)- These are the building blocks for each DNA strand. These are combined to form new strands of the selected DNA.
The Steps of Thermal Cycling-
- Initial step: 95°C for 3 minutes-
- Denature at 95°C for 30 seconds-
- Anneal at 57°C for 30 seconds-
- Extend at 72°C for 30 seconds-
- Final step: 72°C for 3 minutes-
- Final hold: 4°C-
DNA is made up of four types of molecules called nucleotides, designated as A, T, C and G. Base pairing, driven by hydrogen bonding, allows base pairs to stick together.
- Adenine anneals to Thymine
- Thymine anneals to Adenine
- Cytosine anneals to Guanine
- Guanine anneals to Cytosine
Part 4- Base pairing occurs during the Annealing and Primer Extension phases