CP14 Guide Lines Biology A Level Tutor
Activity 6.3
Guide Lines for Core practical 14: DNA Gel Electrophoresis.
Core practical instructions given by Pearson can be found at this link:
https://qualifications.pearson.com/content/dam/pdf/A%20Level/biology-a/2015/teaching-and-learning-
materials/A_level_Biology_A_Core_Practical_14_-_Gel_Electrophoresis.pdf
This CP is a research based activity.
Content:
1. PCR = Polymerase Chain Reaction
2. Restriction endonucleases
3. Electrophoresis
4. Southern blotting and gene probing
1/9
, CP14 Guide Lines Biology A Level Tutor
PCR = Polymerase Chain Reaction
It is cycles of heating and cooling and copying the DNA. All DNA samples must be as
clean as possible and uncontaminated with other DNA otherwise we will have
amplification of the wrong DNA. 35 cycles (few hrs) = 34 billion copies
Materials
Thermocycler
Water
Reaction Buffer (maintain optimum pH for reactions)
MgCl2 (catalyser – increases polymerase productivity)
dNTPs (nucleotides)
Forward and Reverse Primer (Short lengths of artificially synthesised
sequences of DNA bases sequences)
Target DNA
Polymerase Enzyme (Used to join nucleotides after primer initiates
replication)
Steps
1) DNA denaturation: DNA becomes single stranded 94oC or 95 oC for 30 secs
2) Primer Annealing: PCR primers find their complementary targets (sequence) and
attach to them. (This is necessary because DNA Polymerases can only add new
nucleotides to an existing DNA strand – in natural DNA replication this can be a
small piece of RNA). The temperature will depend on the melting point of the
two primers e.g. 50oC or 60oC for 20 seconds
3) Polymerase Extension: Taq polymerase (a DNA polymerase) is producing a
complimentary copy of the DNA (target). At 75 oC for 20 seconds
4) Repeat all steps: then you repeat this cycle for 50 cycles maximum (more than
this the polymerase activity will decrease)
5) Final step: Extra 7 minutes at 72 oC, in order to make sure everything have
finished copying.
To validate: Run the samples on a gel or DNA sequence.
2/9
Activity 6.3
Guide Lines for Core practical 14: DNA Gel Electrophoresis.
Core practical instructions given by Pearson can be found at this link:
https://qualifications.pearson.com/content/dam/pdf/A%20Level/biology-a/2015/teaching-and-learning-
materials/A_level_Biology_A_Core_Practical_14_-_Gel_Electrophoresis.pdf
This CP is a research based activity.
Content:
1. PCR = Polymerase Chain Reaction
2. Restriction endonucleases
3. Electrophoresis
4. Southern blotting and gene probing
1/9
, CP14 Guide Lines Biology A Level Tutor
PCR = Polymerase Chain Reaction
It is cycles of heating and cooling and copying the DNA. All DNA samples must be as
clean as possible and uncontaminated with other DNA otherwise we will have
amplification of the wrong DNA. 35 cycles (few hrs) = 34 billion copies
Materials
Thermocycler
Water
Reaction Buffer (maintain optimum pH for reactions)
MgCl2 (catalyser – increases polymerase productivity)
dNTPs (nucleotides)
Forward and Reverse Primer (Short lengths of artificially synthesised
sequences of DNA bases sequences)
Target DNA
Polymerase Enzyme (Used to join nucleotides after primer initiates
replication)
Steps
1) DNA denaturation: DNA becomes single stranded 94oC or 95 oC for 30 secs
2) Primer Annealing: PCR primers find their complementary targets (sequence) and
attach to them. (This is necessary because DNA Polymerases can only add new
nucleotides to an existing DNA strand – in natural DNA replication this can be a
small piece of RNA). The temperature will depend on the melting point of the
two primers e.g. 50oC or 60oC for 20 seconds
3) Polymerase Extension: Taq polymerase (a DNA polymerase) is producing a
complimentary copy of the DNA (target). At 75 oC for 20 seconds
4) Repeat all steps: then you repeat this cycle for 50 cycles maximum (more than
this the polymerase activity will decrease)
5) Final step: Extra 7 minutes at 72 oC, in order to make sure everything have
finished copying.
To validate: Run the samples on a gel or DNA sequence.
2/9
