BISC 219/F10: RNAi Lab 6: Difference between revisions
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Our goal is to insert our gene of interest into the pL4440 plasmid and transform HT115(DE) bacteria with the newly created plasmid.<br><bR> | Our goal is to insert our gene of interest into the pL4440 plasmid and transform HT115(DE) bacteria with the newly created plasmid.<br><bR> | ||
Restriction enzyme digest of PCR product | |||
== Restriction enzyme digest of PCR product == | |||
Once you have analyzed the agarose gel from last week and determined if your PCR reaction was successful in amplifying your gene of interest you are set to proceed to the next step: Restriction enzyme digest.<br><br> | |||
What do restriction enzymes/endonucleases do? They are enzymes have been isolated from bacteria and cut single or double stranded DNA at specific recognition sequences. These recognition sequences are often palindromic (read the same forwards and backwards). The purpose of these enzymes in bacteria is to eliminate foreign DNA that enters the cells (ie bacteriaphage genomes) to protect the host genome. Companies now purify these enzymes from the bacteria and we can use them to manipulate DNA to link different DNA strands together.<br> | |||
<br> | |||
There are two kinds of "cuts" a restriction enzyme can make, either blunt ended or overhangs called "sticky" ends. The blunt ended cuts cause the DNA to have no single stranded "overhangs" that can facilitate base pairing with another strand of complimentary DNA. This makes joining two pieces together harder but it can be done. The "sticky" ends make joining two pieces together with complimentary base pair overhangs much easier to do and is much more likely to happen during a ligation reaction. <br> | |||
<br> | |||
For more information on restriction enzymes you can read the information on [http://en.wikipedia.org/wiki/Restriction_enzymes Wikipedia] or my favorite site for restriction endonuclease information [http://www.neb.com/nebecomm/products/category1.asp?#2 New England Biolabs]. At NEB you can click on the different enzymes and look at the information they have available about the recognition sequence for cutting, the conditions for effectiveness and lots more.<br> | |||
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Purification of samples (remove enzymes) | Purification of samples (remove enzymes) |
Revision as of 11:30, 24 August 2010
Lab 6: Cloning Your Gene of Interest
Plasmids are circular pieces of DNA that can replicate in bacteria but are not part of the bacterial chromosome. Plasmids are generally circular molecules with fewer base pairs of DNA than the chromosome and with certain sequence elements (called the origin or ori) that allow the plasmid to replicate within the bacterial cytoplasm. Many naturally occurring plasmids have been modified for the purposes of using them as research tools. For example, a gene encoding resistance to an antibiotic can be added to a plasmid so that bacteria carrying the plasmid will become antibiotic resistant. This modification allows for selection of cells that carry plasmid DNA. A simplified map of the C. elegans RNAi plasmid is below:
To enable us to make lots and lots of RNA for RNA interference we need to express our gene at high levels. This is done with a specific strain of E. coli called HT115(DE3). The bacteria cells contain the T7 RNA polymerase gene (contained within a stable insertion of a modified lambda prophage λ DE3) under the control of lac operon regulatory elements. This allows expression of T7 polymerase to be controlled by isopropyl-β-D-thiogalactopyranoside (IPTG), a lactose analogue that induces expression of genes under the control of the lac operon o gene. When IPTG is added, the cells will begin to synthesize lots of T7 RNA polymerase. This T7 RNA polymerase can then bind to the T7 promoter sites on the plasmid and begin to synthesize RNA from both T7 RNA polymerase sites. Because the two single strands of RNA are complementary to each other they will form double stranded RNA within the bacterial cell. Additionally, this particular strain is deficient for the RNAaseIII enzyme that degrades double stranded RNA (dsRNA) in the bacterial cell. This allows for the accumulation of dsRNA in the cell and, thus, our ability to induce and RNAi effect! This E. coli strain carries a tetracyclin resistance gene so these cells can be selected on media containing tetracyclin, while the plasmid contains an ampicillin resistance gene that allows only transfomed cells to grow on media containing ampicillin.
Our goal is to insert our gene of interest into the pL4440 plasmid and transform HT115(DE) bacteria with the newly created plasmid.
Restriction enzyme digest of PCR product
Once you have analyzed the agarose gel from last week and determined if your PCR reaction was successful in amplifying your gene of interest you are set to proceed to the next step: Restriction enzyme digest.
What do restriction enzymes/endonucleases do? They are enzymes have been isolated from bacteria and cut single or double stranded DNA at specific recognition sequences. These recognition sequences are often palindromic (read the same forwards and backwards). The purpose of these enzymes in bacteria is to eliminate foreign DNA that enters the cells (ie bacteriaphage genomes) to protect the host genome. Companies now purify these enzymes from the bacteria and we can use them to manipulate DNA to link different DNA strands together.
There are two kinds of "cuts" a restriction enzyme can make, either blunt ended or overhangs called "sticky" ends. The blunt ended cuts cause the DNA to have no single stranded "overhangs" that can facilitate base pairing with another strand of complimentary DNA. This makes joining two pieces together harder but it can be done. The "sticky" ends make joining two pieces together with complimentary base pair overhangs much easier to do and is much more likely to happen during a ligation reaction.
For more information on restriction enzymes you can read the information on Wikipedia or my favorite site for restriction endonuclease information New England Biolabs. At NEB you can click on the different enzymes and look at the information they have available about the recognition sequence for cutting, the conditions for effectiveness and lots more.
Purification of samples (remove enzymes)
Ligation into vector
Transformation into competent cloning cells
RNAi General Information
Media Recipes
Lab 5: Picking your gene to RNAi
Lab 7: Picking your transformant
Lab 8: Plasmid purification and transformation
Lab 9: Induction of bacteria for RNAi
Lab 10: Scoring your worms and RNA purification
Lab 11: RT PCR reactions