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L36-38 Recomb DNA

USCSOM: Biochemistry: L36-38 Recombinant DNA I&II

QuestionAnswer
What is cDNA? DNA derived from copying RNA
Restriction enzymes tend to leave what on the 3' end and what on the 5' end? 3'OH and 5'PO4
What kind of primer is generally used on mRNA in reverse transcriptase? oligo dT; short oligonucleotides of pure T
What kind of gel is used to separate molecules from a few nts up to a few thousand nt in length? Polyacramide gel
What separation technique is used for bases between 100nt to 20,000nt? Agarose gel
What technique is use dto separate fragments up to 1 million bps? pulsed field gel electrophoresis
What kind of stain is used to visualize DNA fragments from electrophoresis? ethidium bromide
What kind of blot test is used to probe specific DNA fragments? Southern blots
What kind of blot test is used to probe specific RNA fragmnts? Northern blots
What kind of blot test uses antibodies to probe proteins? Western blots
What are hybridization arrays? Advantages? micro-dot arrays of short oligonucleotides; allow testing on many mRNAs simultaneously
Describe the Sanger method. requires a primer, DNA polymerase, and a dideoxy terminator; all four ddNTPs are put on a gel and read from the bottom up
What is a vector? host/carrier DNA into which foreign DNA is grafted
What are two types of vectors? plasmids and viruses
What are the characteristics of plasmids as vectors? easy to work with, good for small DNA fragments
Why would you use viruses for a vector? larger amounts of DNA; more efficient at infecting cells
What are YAC Vectors? yeast artificial chromosomes; hold huge amounts of DNA; awkward to work with and maintain
What are BAC vectors? bacterial artificial chromosome; based on E. coli; fragments of 300kb; current vector of choice
What is pUC18? cloning vector used in E. coli; small circular ds DNA that replicates in E. coli
What is an expression vector? designed to insert cDNAs downstream of a bacterial mormoter
How do you screen cDNA libraries? specific antibodies
What is PCR? polymerase chain reaction
How many bps are the primers in PCR? 20bps; long enough for a unique sequence
What is the general bp limit to amply a sequence via PCR? 10 kb
What is a common thermostable DNA polymerase used in PCR? Taq polymerase
What is a single nucleotide polymorphism? single base pair change that does not code for anything
What is a restriction fragment length polymorphism? mutation that creates or abolishes the recognition site for a restriction enzyme
Created by: jlellerm