RecombinantDNATest2

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RecombinantDNATest2
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2014-12-12 15:25:43
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RecombinantDNATest2
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  1. What is the overall goal of Labs 9-12?
    • 9: RE digest (HindIII and BamHI) of pAMP and pKAN plasmids, then ligation
    • 10: Transformation of E. coli with recombinant DNA using classical method (CaCl2 + heat schock)
    • 11: Replica plating to ID dual-resistance colonies
    • 12: Purification of dual-resistance colonies and determination of which fragments are responsible
  2. How many E. coli cells can be generated from a single cell overnight?
    10^6
  3. Why is E. coli commonly used in labs?
    • Easy to work with
    • Typically nonpathogenic
    • Inexpensive
    • Easily transformed
  4. What is a competent cell?
    • Bacteria that has been treated (made competent) to take up DNA
    • *NOTE - DNA is very hydrophilic and won't normally pass through a cell membrane
    • AKA induced transformation
  5. Describe the classical method of induced transformation
    • CaCL2 + heat shock
    • Rapidly dividing cells have pores in their membrane
    • DNA is suspended in solution of COLD Calcium ion
    • DNA forced into cells by incubating cells and DNA together on ice (causing DNA to bind to surface), briefly heat shocking them (42C) (causing a few of the bound DNA molecules to be taken in by the cell), then putting them back on ice
  6. What are the four phases of bacterial growth w/ description, and how does this apply to competency?
    • Lag phase: little growth, activation of genes
    • Log phase: rapid growth
    • Stationary phase: 
    • Death phase: 
    • *NOTE- competence occurs late in the log phase of browth
  7. During experiment 10 how did we verify that the E. coli was in log phase?
    • Check optimal density (OD) of LB + E. coli at 550nm
    • Should be between 0.3 and 0.4
  8. Which forms of DNA are most readily transformed?
    • Supercoiled DNA has TE of ~10^6
    • Relaxed circular (nicked/covalently closed) has TE of ~10^4
    • Linear DNA has TE of ~10^2
  9. How is TE measured? Describe the calculation
    • CFU/ug of DNA
  10. Why did we allow a 1 hour recovery period after transformation in experiment 10?
    • Kanomycin inhibits protein synthesis
    • The 1 hour recovery period allow expression of Kan resistance gene product before exposure to the plate
  11. Lab 10 - What are satellite colonies?  More on AMP or KAN? why?
    • Satellite colonies are not resistant, but reside in a "zone of resistance" formed by a resistant colony
    • This is seen more frequently on LB-AMP, where cells live until wall replication occurs
    • on LB-KAN unresistant cells will have already died
  12. What is the ENCODE project? What results were found?
    • Encyclopedia of DNA Elements
    • Follow up to HGP, an effort to catalog all functional elements and regulatory elements in the human genome
    • Results: 1.2% of genome codes for exons of protein-coding genes
    • 80% is functional (junk DNA not a thing)
    • 76% is transcribed to RNA
    • 5% is conserved across all mammals (4x higher than 1.2% protein coding region)
  13. Describe the number of genes / what they code for as determined by ENCODE
    • 20687 protein coding genes
    • 18400 RNA genes (no protein)
    • (8800 small RNAs, 9600 large RNAs (>200nt))
    • 11224 pseudogenes
  14. What is alternative splicing?
    There are ~2.53 protein coding variants per locus (range between 1 and 10)
  15. What important RNA breakthrough was discovered with C. elegans
    • sense RNA and antisense RNA worked similarly to suppress genes (no dramatic difference)
    • dsRNA was 10x more potent as silencing trigger (RNA interference)
    • dsRNA can be "fed" to C. elegans by causing expression in E. coli that they eat
  16. What is miRNA? How does it work?
    • microRNA: genomically encoded small non-coding RNA that help regulate gene expression
    • imp. to early development
    • inhibit translation of mRNA bearing complementary target sequence
    • Method
    • miRNA folds in on itself to form dsRNA
    • dicer (RNAse) trims and cuts this dsRNA
    • each strand incorporated into RISC
    • RISC finds the complementary mRNA and cleaves dsRNA to ssRNA, inhibiting translation
  17. Describe the Sanger Sequencing method (early 1980s)
    • REQ - recomb plasmid, DNA pol, 1+ primer, all 4 DNA building blocks, 1 tagged ddNTP
    • 4 reactions, each contains all 4 dNTPs and 1 ddNTP which is tagged with a radioactive tracer
    • *NOTE- dd = dideoxy
    • ddNTPs prevent further replication (chain terminator)
    • Fragment to be sequenced must be cloned into a plasmid
    • Billions of molecules will result in termination at various points in the sequence (A rxn 21, 24, 26; T rxn 23, 29, 33, etc)
    • **NOTE - the A reaction is showing that the termination point is a T (the A was added)
    • To determination the points of termination, each tube is placed on a HIGH RESOLUTION GEL (this as paper, very long)
    • *NOTE - this will allow separation by DNA fragments to a single bp
    • Place X-Ray film over gel overnight, silver is displaced by radioactive trackers
    • Visualize film on lightbox and read upward for best results
  18. What was the major advancement in the Sanger sequencing method in 1988 w/ name? How is data presented?
    • Automated DNA sequencing
    • 4 fluorescent labels (RYBG) rather than a single radioactive tag
    • Bases read by a laser/detector rather than by humans
    • Only a single lane is required, and as the fragment runs off the end of the gel the laser captures the information!!!
    • Data presented in an electropherogram
  19. Describe capillary gel electrophoresis. Advantages?
    • Tubes are filled w/ matrix (like a column)
    • Mostly completed by robots
    • Up to 384 tubes in CE by 2003
    • Advantages: no gel pouring, increased # of runs/day, autoloading, robot completion
    • *NOTE - no longer used, due to newer tech
  20. What is the biggest advantage for next generation sequencing?
    • No bacterial clones
    • No vectors, no ligase, no transformation
    • *NOTE - isolate genomic DNA, fragment the DNA, add dsDNA tag to each end
  21. Describe emulsion PCR + pyrosequencing
    • fragmented DNA has dsDNA tag added to each end
    • suspended beads (single bead in water droplet) have attached primers that are complementary to the DNA tags
    • PCR occurs within each droplet (only 15% successful, but others do not interfere)
    • Denature DNA -> anneal to bead -> extension of DNA fragment -> denature 1 strand from beat -> repeat)
    • break emulsions and deposit beads onto picotiter plate (3.6m wells, only ~1m have bead w/ single frag)
    • **NOTE - each company reads the results differently
    • company 1 (454 sequencing)
    • pyrosequencing
    • mix DNA, DNA pol, ATP sulfurylase (enz), APS (subs), luciferase (enz), luciferin (subs), and apyrase (degrades nucleotides)
    • 1 rxn creates ATP, other rxn creates light
    • integration of nucleotide releases PPi
    • PPi + APS -> ATP, ATP + luciferase -> light
    • nucelotides added one at a time every 8 minutes until light is seen, then repeated
    • *NOTE - apyrase is what degrades the free nucleotides in between each treatment
    • Light captured by camera, height = related to # of nucleotide added
    • *NOTE - only 400bp per rxn, but 1 million+ rxns
  22. Describe bridge amplification
    • similar to pyrosequencing
    • amplification on "flow cell" (flat) not on a bead
    • nucleotides have 4 fluorescent tags
    • Flow cell imaged after each addition of 4 nucleotides
    • Molecules form "bridges" as they are amplified
    • software keeps track of each signal
    • *NOTE - 75bp per rxn, 300 million rxns
  23. Describe the ABI SOLiD system
    • very different from other 2 next gen sequencing techniques
    • 50bp per rxn, >1 billion simultaneous rxn
    • high accuracy (99.94)
  24. Describe how next generation sequencing techniques are creating breakthroughs in medicine (specific article)
    • Researchers closely examined the genome of 158 people with brain malformations
    • Using sensitive sequencing techniques they covered a depth (how many times each sequence is read) of 300x (more than 5x the normal depth)
    • This allowed them to ID mutations that would have otherwise been dismissed as read errors!!!
    • Only deep sequencing allowed them to find these mutations! (Sanger would've missed them, and it is still the "golden standard")
  25. What is the CRISPR-Cas9 system? (general)
    • CRISPR - clustered regualrly interspaced short palindromic repeats
    • Cas - CRISPR associated proteins
    • Cas9 is a nuclease
    • First discovered in bacteria, it functions as a defense against foreign DNA (we use Type II)
    • It is now a unique tool that allows insertion of DNA askjdnknfa2f
  26. Describe the spacer-repeat construct RE: CRISPR
    During invasion (viral or plasmid) a portion of the previous invader's DNA is inserted as spacers in the repeat-spacer-repeat-spacer construct
  27. What is the function of CRISPR/Cas in bacteria?
    • The CRISPR/Cas system targets foreign DNA with a short complementary ssRNA
    • This localizes the Cas9 nuclease and causes a dsbreak, resulting in the silencing of that DNA sequence
    • *NOTE - Protospacer Adjacent Motif (PAM) must be present for the binding of the complex
  28. What are the 2 major breakthroughs used in CRISPR/Cas?
    • The genes for crRNA and tracrRNA can be JOINED creating a single GuideRNA which is functional!!!! (simply add a loop to bind them together)
    • This works in eukaryotic cells - and the ds break can be repaired (or inserted :) )
    • Only the target sequence need be changed to target specific genes!
  29. How can CRISPR/Cas be used with a library of cells?
    Genes can be "knocked out" to determine function
  30. What are the requirements for a functional plasmid (re: experiment 12)
    • Have AmpR and KanR
    • Have origin of replication (2 is ok)
    • Hind to Hind, Bam to Bam
    • Must be circular
    • Limit 4 peices (large size prevents entry to E. coli)
    • Same pieces cannot be "next door neighbors"
  31. What are the two names for the "special plasmids" from experiment 12?
    • Super plasmid (all 4)
    • simple recombinant (only 2 required)
  32. Why do multiple lanes appear when viewing the lanes for experiment 12? What is the important one?
    • Supercoiled (will travel furthest, only one to compare)
    • nicked
    • multimer
  33. What can't 2 fragments be "next door"?
    • Complementary strands will occur both next to and across from a given strand
    • this causes a loop to form, and replication is unlikely
  34. Why do we record 3 values from the nanodrop?
    • Helps to verify that we have DNA, not protein
    • Concentration [ng/uL]
    • 260/280 ~ 1.8 for DNA
    • 260/230 ~ 2-2.2 for nucleic acids
  35. For the C. glutamicum RE digests, why were the products passed over a column between digests?
    To remove remaining RE and RE buffer that would interfere with the next digest.

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