dna isolationdna manipulation and molecular cloning
rt pcr qpcr
site directed mutagenesis
fluorescent labelsprobes
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Vrije Universiteit Amsterdam (VU)
Minor Biomolecular Sciences And Neuroscience
Experimental Cell Biology 2
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Experimental Cell Biology II: Basic Techniques
Cell biology techniques: DNA manipulation and molecular cloning
• DNA/RNA techniques After the DNA is purified it can also be used to
• Protein techniques manipulate or clone the DNA.
• Knock-out To manipulate the DNA, the DNA can be split by
• Knock-down with RNAi enzymes to cut a specific sequence out of the
• CRISPR-Cas DNA. Restriction endonucleases are used to cut a
specific sequence out of the DNA at the specific
Cell tissues can be broken up by: restriction sites, these are about 4-8 bases long.
• Grinder or blender To cut the DNA, all restriction enzymes make
• Osmotic shock two incisions, once through each sugar-
• High pressure phosphate backbone of each strand and one of
• Detergent/enzyme treatment the DNA double helix. Once the piece is out of the
DNA, it can be used to insert inside another DNA
DNA techniques: sequence, which also has to be cut open. After
• Isolation putting two different sequences together in
• Manipulation/cloning solution, DNA ligase can be used to glue the ends
• Amplification of all the fragments together. After inserting the
• Investigation recombinant DNA into a host cell, the DNA can
be transcribed into mRNA and translated into a
DNA isolation protein by RNA polymerase.
To isolate DNA, a cell extract is prepared
during a process called lysis, where the To clone the recombinant DNA, the DNA requires
nucleus and the cell are broken open, thus a cloning vector. The DNA is inserted in the host
releasing the DNA. To free the DNA from its organism/cell. A polymerase chain reaction
proteins and other cell components, they are (PCR) is used to replicate the recombinant DNA
dissolved using enzymes such as Proteinase K sequence inside the host cell.
and detergents or an organic solvent. During
the precipitation step, the DNA is freed from Polymerase chain reaction
cellular debris. It involves using sodium ions to For the PCR, you need the DNA fragment,
neutralize the negative charge in DNA nucleotides, primers, buffer, and DNA
molecules (making them more stable). After polymerase. During the first step of the PCR, the
this, alcohol, ethanol or isopropanol is added to two strands of DNA are separated at a high
complete the precipitation and free the DNA. temperature, this is called nucleic acid
The DNA is then purified in a buffer solution or denaturation. In the second step, annealing, the
alcohol, which removes all the remaining temperature is lowered so that the primers can
cellular debris and unwanted material. bind to the DNA. After this, an intermediate
• There are kits available that often use a temperature is used so that the DNA polymerase
porous material for specific binding of DNA can make a new strand, this step is called
extension. These cycles are repeated 20-40
Agarose gel electrophoresis times.
This technique uses porous gel to separate
DNA fragments. The fragments are separated
by applying an electric field to move the
charged molecules through an agarose matrix,
negatively charged DNA moves to the positive
side of the electric field. The fragments are
separated according to size. Smaller DNA
fragments move further in the gel. The Reverse transcription PCR (RT-PCR)
separated DNA may be viewed with stain This is a modified version of PCR that amplifies
(DNA-binding dye, ethidium bromide), most RNA to investigate the mRNA in the cell. For
commonly under UV light, they can also be this, a reverse transcriptase is added to
extracted from the gel by cutting the wanted synthesize DNA from mRNA, after this a normal
DNA out. PCR reaction is performed.
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