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DNA Cloning Experiment: Isolation and Restriction Digestion of DNA

General Aim of DNA Cloning Experiment

 

• DNA extraction from a culture of Saccharomyces Cerevisiae, followed by restriction enzyme digestion using restriction enzyme EcoRI. 
• Restriction digestion and dephosphorylation are also applied on plasmid pUC19.

 

Method of DNA Cloning Experiment

DNA isolation using purification columns in a gene cloning experiment. DNA digestion using restriction enzyme EcoRI.

Theoretical Background/Context

• DNA extraction from yeast ‘Saccharomyces cerevisiae’ is done using purification columns. 
• A Restriction enzyme (EcoRI) is used to cut the DNA fragment of interest from the extracted DNA through restriction enzyme digestion.
• Restriction enzymes are present in bacteria. They are named according to the bacteria from which they are extracted. 
• They protect bacteria by foreign DNA digestion at specific sequences EcoRI recognizes the 6 bp sequence 5’ GAATTC 3’ and makes a staggered cut between the G and A creating sticky ends.

5’ GAATTC 3’     after         5’ G         AATTC 3’

3’ CTTAAG 5’   restriction   3’ CTTAA       G 5’   

• Now that the yeast DNA fragment of interest is cut, multiple copies of it are to be created through cloning DNA inside a living cell.
• Therefore, you combine it with a vector. Vectors are segments of DNA into which target genes can be inserted. 
• Vectors can then be introduced into a living cell, like a bacterium, where it will continue to divide. 
• This multiplies the DNA fragment and makes it much easier to study. 
• The vector you will be using is a plasmid called ‘pUC19’. It will be cut using the same restriction enzyme ‘EcoRI’. 
• Plasmids are small circular double stranded DNA that can replicate independently of the chromosomal DNA in bacteria. 
• Using plasmids allows the isolation, analysis, and manipulation of fragments of DNA. 
• The 5’ phosphate groups of DNA are removed from the plasmid after its restriction using alkaline phosphatase. 
• This should prevent vector recircularization during the ligation reaction later on. 

Principle of Work

At DNA Cloning Experiment:

• Cells are lysed at first to break down the cell wall and extract DNA into the solution. 
• The solution is then treated with proteinase K and RNase A, which remove proteins and RNA in the sample, respectively. 
• The sample is then loaded onto a DNA Purification column containing a silica membrane. 
• The genomic DNA will bind to the membrane, whereas the impurities will be removed upon washing the column with wash buffers.
•  The genomic DNA is then eluted from the column under low ionic strength using the elution buffer in the final step of the DNA Cloning Experiment.