Learning Goal - DNA Replication - Polymerase chain reaction and gel electrophoresis as tools for amplifying and separating DNA
3 important questions on Learning Goal - DNA Replication - Polymerase chain reaction and gel electrophoresis as tools for amplifying and separating DNA
Outline the process of PCR, including the use of primers, temperature changes and Taq polymerase.
Step 1: Increase temperature to break hydrogen bonds between the base pairs of the 2 strands of DNA in a DNA molecule --> unwind the double-stranded helix to expose individual strands (around 95 degrees).
Step 2: Reduce the temperature to allow DNA primers to stick to the exposed DNA strands, just before the target gene (around 40 degrees).
Step 3: Utilise Taq polymerase (bacterial polymerase) to replicate the target base sequences, and adjust the temperature to around 72 degrees, optimal for Taq polymerase activity.
Step 4: Cycle repeats.
Step 2: Reduce the temperature to allow DNA primers to stick to the exposed DNA strands, just before the target gene (around 40 degrees).
Step 3: Utilise Taq polymerase (bacterial polymerase) to replicate the target base sequences, and adjust the temperature to around 72 degrees, optimal for Taq polymerase activity.
Step 4: Cycle repeats.
Deduce the number and relative size of the DNA fragments from the number of bands in an electrophoresis gel.
Number of different-sized bands after staining represents the number of different-sized DNA fragments in the molecule. The small pores in the gel act as a molecular sieve, allowing smaller fragments to move faster through the gel and larger fragments to move slower, separating them. Fragments of the same sizes move to the same position on the gel (are on the same line).
Give me details of the type of stains/gel typically used for DNA electrophoresis.
Gel: Agarose Gel or Polyacrylamide Gel (PAG)
Stains: Methylene Blue or Ethidium Bromide
Stains: Methylene Blue or Ethidium Bromide
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