Cloning-DNA Isolation and Restriction Digestion | PraxiLabs

Cloning-DNA Isolation and Restriction Digestion Virtual Lab Simulation

Biology | Biochemistry | Genetics | Microbiology



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General Aim

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

Method

DNA isolation using purification columns. Restriction digestion of DNA using restriction enzyme EcoRI.

Learning Objectives (ILO’s)

  • To Apply the steps of DNA extraction from a culture of Saccharomyces Cerevisiae.

  • To execute proper restriction enzyme digestion of DNA.

  • To understand the role of the devices, the reagents and the enzymes used in the process of DNA extraction.

  • To implement proper storage of samples.

  • To understand the role of the devices, the reagents and the enzymes used in the process of DNA extraction.

  • To implement proper storage of samples.

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. Restriction enzymes are present in bacteria. They are named according to the bacteria from which they are extracted. They protect bacteria by digesting foreign DNA 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 replication 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

Cells are lysed at first to break down cell wall and extract DNA into the solution. Solution is then treated with proteinase K and RNase A, which remove proteins and RNA in 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. 


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