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Lecture 8

Brandl Lecture 8

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Biochemistry 2280A
Eric Ball

Biochem Lecture 8 - Nov. 24th Case Study (con’t.) Purifying the PCR Product: • DNA molecules are most easily separated base on difference in length (# of base pairs) • when places in an electric field at pH7, DNA will migrate towards the positive pole because the DNA phosphate backbone is negatively charged in neural pH Gel Electrophoresis: • used to identify and resolve DNA molecules • DNA is sieved through a matrix of agarose (or acrylamide) • DNA from each PCR reaction is loaded into a well in the agarose gel • the gel is in a chamber in which an electric field can be applied • when an electric current is passed through the chamber, DNA fragments are pulled through the agarose matrix and move towards the positive pole • agarose contains small pores that the DNA must move through • smaller DNA fragments move faster than large DNA fragments as they fit through the pores easier Detection of the DNA in the Gel: ← 1. Stain with ethidium bromide: • Eth Br fluoresces red under UV light when bound to DNA • can then cut out and isolate “glowing” DNA • can detect about 100ng of DNA ← 2. Autoradiography • radioactively label the 5’ ends of your DNA fragments using polynu- cleotide kinase and [32P]-ATP • results in radioactive DNA • expose your gel to an x-ray film • very sensitive • used for very small amounts of DNA What Do We Use as a Template for the PCR?: • Question: If our goal is to express YFG (in humans) would we use genomic DNA as the template? • Answer: No, unless you can find a human gene that lack introns • Introns are a problem in expressing eucaryotic genes in E. coli because bacteria do not possess the machinery to splice them out cDNA: • cDNA is a replicate DNA copy of mRNA (c=complementary) • cDNA does not contain introns as it is based off of RNA • each cDNA encodes one gene • the cDNA can be used in PCR Making cDNA: • Figure 10.13 • isolate and purify mRNA from cell • hybridize a poly(t) primer to the a-tail of the mRNA • add reverse transcriptase (viral enzyme) and dNTPs • makes a DNA copy • essentially the reverse of transcription • creates a double stranded DNA-RNA helix • treat with alkali to degrade RNA • results in single stranded DNA • single stranded cDNA can be used in a PCR reaction Source of the mRNA: • you need to identify a tissue or human cell line in which YFG is expressed • hybridization/Northern Blot • hybridization is a powerful tool to identify nucleic acid sequences of interest • hybridization experiments are generally done after transferring the DNA or RNA to a nitrocellulose membrane • Southern Blot: single stand DNA to single strand DNA • Northern Blot: single strand DNA to RNA Example of a Northern Blot: • identify w
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