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L36-38 Recomb DNA
USCSOM: Biochemistry: L36-38 Recombinant DNA I&II
| Question | Answer |
|---|---|
| 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
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