BIMM 100 techniques sheet

BIMM 100 techniques sheet - Technique What does it look for...

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Technique What does it look for Procedure Plaque Assay Allows to count for viruses in cells Have a confluent layer of host cells on a plate and add a viral suspension Then observe for plaques or areas of dead cells. 1 plaque = 1 virus affecting that area Making a clone Allows for making of a plasmid in order to transfect cells and test the proteins Use restriction enzymes to cleave DNA at desired site. The sticky ends can then bind to the sticky ends of a host vector. The vector has three main parts: selection area, replication origin and specific site for DNA entry. Use DNA ligase to ligate the DNA in the vector. Uses 2 ATP. Then allow for transformation into cells and allow for plasmid replication. Forms a colony of cells resistant due to the selection area. cDNA library Can use the library to find a clone of interest Start with mRNA and make single stranded DNA add a complementary strand and now have cDNA. Can take this and can digest with restriction enzymes to add to plasmid. Use the E coli to transform and get cells transfected with the plasmid. Use the cells to produce a group of plasmid clones via replication and the purified phage is the library. PCR Used to amplify a target DNA Start with DNA template, use thermostable DNA polymerase, add RNA Primers and dNTPs. The reaction will repeat and each time the amount of DNA is doubled. Southern Blot Genomic, detects DNA fragments. Is the DNA present? Use restriction enzymes to cleave DNA and electrophorese. Place the gel in NaOH to denature the DNA. Transfer DNA onto a filter. Allow for labled flourecent DNA to hybridize to complementary strands. Wash and use xray film to find the dark bands of hybridized DNA which will show the gene is present.
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Norhter blots Test for expression, want to test for specific mRNA Run all mRNA on a gel. Go to cDNA library and make a probe with fluorescent dNTPs. Blot in alkaline solution so that the mRNA can bind. If it is upregulated,
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BIMM 100 techniques sheet - Technique What does it look for...

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