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| a short (3-60 amino acids long) peptide chain that directs the post-translational transport of a protein. Signal peptides may also be called targeting signals, signal sequences, transit peptides, or localization signals. The amino acid sequences of signal peptides direct proteins (which are synthesized in the cytosol) to certain organelles such as the nucleus, mitochondrial matrix, endoplasmic reticulum, chloroplast, apoplast and peroxisome. Some signal peptides are cleaved from the protein by signal peptidase after the proteins are transported. |
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| A protein signal patch contains information to send a given protein to the indicated location in the cell. It is made up of amino acid residues that are distant to one another in the primary sequence, but come close to each other in the tertiary structure of the folded protein (see red patch in the diagram). |
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| have an effect for protein trafficking |
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| A biosensor is a device for the detection of an analyte that combines a biological component with a physicochemical detector component.[1] |
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| A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA. The enzyme makes two incisions, one through each of the sugar-phosphate backbones (i.e., each strand) of the double helix without damaging the nitrogenous bases. The term restriction comes from the fact that these enzymes were discovered in E. coli strains that appeared to be restricting the infection by certain bacteriophages. Restriction enzymes therefore are believed to be a mechanism evolved by bacteria to resist viral attack and to help in the removal of viral sequences. They are part of what is called the restriction modification system. |
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| emits light - fluoresence |
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| Fluorescent proteins can do this |
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| Green fluorescent protein |
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GFP - 238 amino acid protein from jelly fish can-like and has a chromophore in the middle - insulation for fluorescence DOES NOT GO INTO THE NUCLEUS |
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a red fluorescent protein RFP from Discosoma coral |
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| how to transfect eukaryotic cells |
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EUKARYOTIC CELLS coat DNA with lipid to fuse into the membrane - a by-chance event that it it will be packaged |
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| where you put the DNA in after you cut in the vector |
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| Molecular cloning refers to the procedure of isolating a defined DNA sequence and obtaining multiple copies of it in vivo. Cloning is frequently employed to amplify DNA fragments containing genes, but it can be used to amplify any DNA sequence such as promoters, non-coding sequences, chemically synthesised oligonucleotides and randomly fragmented DNA. Cloning is utilized in a wide array of biological experiments and technological applications such as large scale protein production. |
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| Gene expression is the process in which the inheritable information in a gene, such as the DNA sequence, is made into a functional gene product, such as protein or RNA. |
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| Transfection of Cultured Cells |
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| Transfection describes the introduction of foreign material into eukaryotic cells using a virus vector or other means of transfer. |
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| transformation is preferred to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells such as fungi, algae and plants. |
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proteins that will fluoresce when they hit the light this is used as a reporter of expression |
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DNA is palendromic the enzymes cut at sequences 4-6 nucleotides long they make sticky ends ligase restores the phosphodiester bonds |
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water sticky ends ATP buffer ( .25 to 1 in concentration) to attach t4 ligase sticky ends use less ligase than the blunt ends |
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| Annealing, in genetics, means for DNA or RNA to pair by HYDROGEN BONDS!!! to a complementary sequence, forming a double-stranded polynucleotide. The term is often used to describe the binding of a DNA probe, or the binding of a primer to a DNA strand during a polymerase chain reaction (PCR). |
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ligase works over a broad temperature range 16 degrees C is the optimal range |
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The polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify (i.e., replicate) a piece of DNA by in vitro enzymatic replication. DNA template is exponentially amplified. |
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| employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme derived from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, using single-stranded DNA as template and DNA oligonucleotides (also called DNA primers) required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These different temperature steps are necessary to bring about physical separation of the strands in a DNA double helix (DNA melting), and permit DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The power and selectivity of PCR are primarily due to selecting primers that are highly complementary to the DNA region targeted for amplification, and to the thermal cycling conditions used. Developed in 1983 by Kary Mullis,[1] PCR is now a common and often indispensable technique used in m |
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DNA template dNTP primers buffer (MG++) Thermo-stable DNA polymerase |
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| A cloning vector is a small DNA vehicle into which a foreign DNA fragment can be inserted. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with the same restriction enzyme, then ligating the fragments together. |
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| An expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce and express a specific gene into a target cell. Expression vector allows production of large amounts of stable mRNA. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular transcription and translation machinery. The plasmid is engineered such that it contains a highly active promoter which causes the production of large amounts of mRNA. |
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| In molecular biology, transformation is the genetic alteration of a cell resulting from the uptake and expression of foreign genetic material (DNA). Separate terms are used for genetic alterations resulting from introduction of DNA by viruses ("transduction") or by cell-cell contact between bacteria ("conjugation"). Transformation of animal cells is usually called transfection. |
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| a cloning vector must have these to AMPLIFY |
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replication origin a selectiion marker a cloning site |
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| A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (usually 20+) restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique. In other words, they only occur once within that particular plasmid. MCSs are commonly used during procedures involving molecular cloning or subcloning. Extremely useful in biotechnology, bioengineering, and molecular genetics, MCSs let a biotechnologist insert a piece of DNA or several pieces of DNA into the region of the MCS. This can be used to create transgenic organisms, also known as genetically modified organisms (GMOs) |
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| catalyzes and guides the unknotting of DNA |
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| putting the DNA in a certain way |
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| treat bacteria to make the wall permeable |
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| mix bacteria with dna and put under hig voltage to open the bacterial wall |
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| transformation efficiency |
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| 10^7 or 10^8 cfu /microgram is good efficiency |
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this plus heat chock could transfect E.Coli with phage or plasmid DNA you don't want the bacteria to repair the sequence though |
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5' has the phosphate 3' has the sugar |
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| when a vector is cut bluntly.. |
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| no phosphate or hydroxy group for primers that aremade chemically do not have these |
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can repair the fact that the bacteria does not have a phosphate or sugar group match or you can treat with DNA phosphataseto remove the phosphate groups so that it can never ligate itsef. it can ligate when you add a piece of DNA with the phosphate group on it. |
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| Intracellular transport of protein |
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| must rely on signals to determine where they go |
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| transfection of eukaryotic cells |
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| once you have the signals AND put it into an expression vector, you cantell it where to go |
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| made and transported into the nucleus and mitochondria |
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| transportedto the secretory vesicles |
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| TAE buffer is a buffer solution used in agarose electrophoresis, typically for the separation of nucleic acids such as DNA and RNA. It is made up of Tris-acetate buffer, usually at pH 8.0, and EDTA, which sequesters divalent cations. TAE has a lower buffer capacity than TBE, but linear, double stranded DNA runs faster in TAE. |
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| a molecular weight marker for gel electrophoresis |
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| a staining dye that is fluorescent to document gel electrophoresis |
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