BIOTEC LAB E2 ALL C

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shockwave
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BIOTEC LAB E2 ALL C
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2011-11-17 09:22:21
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BIOTEC LAB E2 ALL C
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  1. CLASSIC PROCEDURE FOR PREPAIRING COMPETENT CELLS WAS WRITTEN BY WHO AND WHEN?
    • 1970
    • MANDEL & HIGA
  2. By what factor is the classic procedure more or less efficient than colony transformation?
    colony protocol:5 x 103 to 5 X 104 colonies per µg of plasmid.

    THE CLASSIC transformation efficiencies are 5 x 104 to 5 X 106 colonies per µg of plasmid.

    colony protocol (C5) is up to 200 times less than those of the classic protocol (C8).
  3. WHAT 2 PROBLEM DOES CLASSIC SOLVE THAT COLONIAL HAS.
    1. LIGATES PLASMA DNA -COMPOSED OF LINEAR OR RELAX -TRANSFORm LESS EFFICENTLY THAN SUPERCOILED PLASMID.

    2. DUEL SELECTION WITH KAN & AMP IS UNFORGIVING AND KILLS A PERCENTAGE OF TRANSFORMANTS CONTAINING BOTH RESISTANCE GENES.
  4. WHAT THE HELL DOES SEASONING MEANS?
    ADDING CaCl TO HELP DRY OR REMOVE WATER FROM THE PELLET.
  5. SEASONING THE CELLS INCREASES WHAT BY HOW MUCH?
    TRANSFORMATION BY 5 TO TEN FOLD.
  6. DIFFERENCE BTWN COLONY VS CLASSICAL WHEN IT COMES TO STARTING OFF
    • COLONY IS STARTED BY USING ECOLI IN A VARIETY OF GROWTH STAGES AND MIXED WITH PLASMIN pAMP.
    • CLASSICAL (NEWER) IS STARTED WITH MID-LOG CELLS THAT HAVE ALREADY BE TRANSFORMED.
  7. FORMULA FOR TOTAL MASS OF pAMP
    • MASS= VOLUME * CONCENTRATIONIN THE EXPERIEMTN IT WAS
    • 10 uL * 5ug/uL= .05ug
  8. FRACTION OF CELL SUSPENSION SPREAD ONTO “A” LB/amp PLATE...HOW?
    FRACTION SPREAD = volume suspension spread/total volume suspension.
  9. “A” LB/amp =100µl/1000µl= 1/10= .10µl was the volume of the fraction of cell suspension spread.
  10. MASS OF PLASMID SPREAD ONTO “A” LB/amp PLATE. FORMULA.
    • MASS PLASMID SPREAD
    • = total mass plasmid x fraction spread
    • .05µg of pAMP (and pKAN) was the total mass.
    • .10µl was the volume of the fraction of cell suspension spread.
    • .05µg * 0.10µl = .005 µg/µl was the amount of the mass plasmid spread per pAMP (and pKAN).
  11. NUMBER OF COLONIES PER µg OF pAMP & pKAN. WHAT DO YOU USE?
    TRANSFORMATION EFFICIENCY =

    • colonies observed
    • -----------------------
    • mass plasmid spread

    pAMP =4,212 CFU/.005 µg = 8.424 X 105 colonies/ µg.
  12. HOW DOES ONE PREPAIR COMPETENT CELLS?
    • START WITH MID LOG
    • SPIN TO PELLET
    • SEASON WITH CaCl ICE BATH FOR 20
    • SPIN AND DRAIN
    • SEASON WITH CaCl
    • VORTEX AND STORE.
  13. HOW DOES ONE TRANSFORM E. COLI WITH RECOMBANT PAMP/PKAN PLASMIDS?
    • COMPENTENT CELL TO +PAMP THEN ICE
    • ADD LIGATED TO SAME TUBE THEN ICE 20
    • HEAT SHOCK 42 THEN ICE
    • LB TO TUBE (ALL) 37 FOR 50
    • SPREAD ON PLATES
    • INCUBATE 37 12-24HRS.
  14. THE SOUTHERN BOLT TECH RELIES ON WHAT?
    HYDROGEN BONDSTHAT HOLD TOGTHER COMPLEMENTARY BASE PAIRS IN DOUBLE STRANDED DNA.
  15. AT TEMPS ABOVE ---- OR pH ABOVE ----- WHAT HAPPENS TO HYDROGEN BONDS?
    • ABOVE 90
    • pH 10.5
    • HYDROGEN BONDS ARE DISRUPTED AND COMPLEMENTARY STRANDS DENATURE INTO SINGLE STRANDS.
  16. 3 SOURCES OF SINGLE STRANDED DNA HYBRIDIZATION PROBES.
    • RESTRICTION FRAGMENT
    • PLASMID CONTAING CLONED SEQUENCE
    • SYNTHETIC OLIGONUCLEOTIDE
  17. NAME 3 WAYS TO VISUALIZE A PROBE
    • RADIOACTIVE
    • COLORMETRIC
    • CHEMILUMINESCENT
  18. NAME THE BASIC STEPS OF THE S BLOT
    • RESTRICTION -->GET FRAGMENTS
    • RUN GEL OF LAMDA DNA
    • STAIN GEL, RINE, UV VIEW &PHOTO
    • DENATURE (DOUBLE RINSE)
    • BLOT
    • WASH & BAKE NYLON MEMBRANE
    • PREHYBRIDZE & HYBRIDIZE
    • WASH & DEVELOP
  19. AT THE BEGINING OF A TYPICAL SOUTHERN HYBRIDIZATION-------- OF GENOMIC DNA IS CUT WITH ONE OR MORE ------- -------.
    • 10ug
    • RESTRICTION ENZYMES
  20. A --------- WITH A ----BP RECOGNITION SEQUENCE STATICALLY CUTS ONCE EVERY ----- BP(X).
    • ENDONUCLEASE
    • 6
    • 4,096 (46)
  21. E.COLI HAS HOW MANY BP IN GENOME AND HOW MANY RESTRICTION FRAGMENTS?
    • 4 MILLION BP
    • 977 RESTRICTION FRAGMENTS
  22. IN SOUTHERN BLOTTING YOU REALLY DON'T GET SMEARS. WHY?
    SMALL SIZE OF LAMDA GENOME (48,502) AND THE LARGE DNA LOAD (6ug) ENSURE REPRODUCIBLE RESULTS AND SHORT HYBRIDIZATION TIMES.
  23. SOUTHERN BLOTTING IS SO SENSITIVE THAT.....
    ITS CAPABLE OF DETECTINGH A RESTRICTION FRAGMENT THAT IS PRESENT ONLY ONCE IN THE HUMAN GENOME.
  24. HOW MANY BP SND RESTRICTION FRAGMENTS IN HUMAN GENOME?
    • 3.5 BILLION
    • 854,492 FRAGMENTS
  25. THREE METHODS ARE COMMONLY USED TO TRANSFER NUCLEIC ACIDS FROM GELS TO NYLON NAME THEM.
    1. CAPILLARY..SOUTHERN'S IDEA

    2. ELECTROPHORETIC..2-3 HRS USING CURRENT, BUT BUFFER MUST BE CYCLED TO BE COOL. BEST FOR POLYACRYLAMIDE.

    3. VACUUM FASTEST. MOST EFFICENT. VACUUM FROM THE BOTTOM.
  26. it’s flammable. Nucleic acids are draw to it via hydrophobic interactions. WHO AM I?
    Nitrocellulose MEMBRANE
  27. WHAT MEMBRANE BINDING IS REVESABLE AND WHAT ABOUT THE OTHER?
    NITROCELLULOSE.

    NYLON MEMBRANES BIND NUCLIC ACIDS IRREVESTABLE UNDER LOW IONIC STRENGTH.
  28. NYLON CAN UNDGO MANY HYBRIDIZATION WHERE AS NITOCELLULOSE CAN'T.

    TRUE?
    TRUE.
  29. THE SOLE DISADVANTAGE OF NYLON MEMEBRANE IS...
    SOMETIMES GIVE HIGHER LEVELS OF BACKGROUND, ESPICALLY WITH RNA PROBES.
  30. WHAT FILTER PAPER SPEC IS REFERENCED IN THE TEXT?
    WHATMAN 3mm
    • P = PAPER TOWELS (FILTER IS BENEATH)
    • A = AGAROSE GEL
    • W =WICK
    • N = NYLON MEMBRANE
    • S = HIGH SALT SOLUTION
  31. WHAT TYPE OF BUFFER IS PLACE IN THE TRAY?
    500 ml OF 10x SSC BUFFER
  32. WHEN YOU WASH AND BAKE AFTER BLOTTING WHAT IS THE SOLUTION THATS USED?
    WASH WITH 100ml OF 2X SSC BUFFER FOR ONE MINUTE, THEN BAKE AT 70 FOR 30MNS OR LONGER.
  33. DENATURE AND NEUTRALIZE DNA STEP...WHAT IS IT?
    • STEP BEFORE YOU BLOT.
    • DENATURE FOR 15MNS TWICE
    • THEN
    • NEUTRALIZE FOR 15MNS TWICE
  34. NAME THE STEPS AFTER THE BLOTTING
    • WASH & BAKE
    • PREHYBRIDIZE & HYBRIDIZE
    • PROBE
  35. HYBRIDIZATION TO EURKARYOTIC GENMOES ARE USUALLY CARRIED OUT AT ---- IN AQUEOUS SOLUTIONS OR AT ----- IN -----------.
    • 68
    • 42
    • 50% FORMAMIDE
  36. HYBRIDIZATION AT LOWER TEMPS IN FORMAMIDE IS LESS DAMAGING TO THE NITROCELLULOSE MEMBRANE BUT......
    TAKES 2-3 TIMES LONGER THAN AT HIGHER TEMPS IN AQUIOUS SOLUTION.
  37. THE RATE OF DNA REASSOCIATIONG INCREASES AS THE THE VOLUME--------
    DECREASES.

    THUS REDUCING THE AMOUNT OF PROBE REQUIRED AND HYBRIDIZIATION TIME.
  38. T OR F?
    THE USE OF A BLOCKING REAGENT IS SOMETIMES OMITTED HEN USING NYLON MEMBRANES BECAUSE THE PROTEIN CAN INTERFER WITH PROBE BINDING.
    TRUE
  39. NON FAT MILK OR BOVINE SERIUM ALBIUMIS USED FOR WHAT IN BLOTTING?
    BLOCKING REAGENT
  40. NAME 3 TYPES OF PROBE DETECTION
    • RADIOIACTIVE...OLD AND DANGEROUS
    • COLORIMETRIC
    • CHEMLLUMINATION
  41. WHAT PROBE USES DIGOXIGENIN OR BIOTIN?
    COLORIMETRIC
  42. WHAT PROBE TYPE USES FILM AND A DARKROOM?
    CHEMILUMINSCENTATION
  43. T OR F?
    WHEN USING RESTRICTION FRAGMENTS AS THE PROBE, YOU SHOULD INCREASE THE STRINGENCY OF THE HYBRIZIDZATION
    NAME THE DETAILS.
    • TRUE
    • 65 AND 5X SSC BUFFER
  44. THIS PLANT STEROID MOLECULE IS HIGHLY ANTIGENTIC AND IS USED AS A PROBE FOR IMMUNE DETECTION.
    DIGOXIGENIN
  45. WHAT PLANTS DOES THE DIGOZIGENIN COME FROM?
    FOXGLOVE & DIGITALIS
  46. HOW DOES ONE PREPAIR DIGIOZIGENIN?
    • IMMUNE SHEEP
    • GET IGg VIA ION EXCAHANGE CHROM.
    • ISLOATE VIA IMMUNOSORPTION (COLUMN)
    • LINKED TO ENZYME ALKALINE PHOSPHATE.
    • YOU HAVE Ab/ENZYME PROBE THAT FORM IMMUNE COMPLEX (PROBE).
  47. ALKALINE PHOSPHATE CATALYZES THE REMOVAL OF A ----- GROUP FROM
    5-BROMO-4 CHLOR-3 INDOLPHOSPHATE
    (X PHOSPHATE). THE RESULTING OXIDATION DIMERIZATION FORMS WHAT?
    • PHOSPHATE
    • INDGO PARCIPATANT
  48. HYDRIDE IONS RELEASED DURING DIMERIZATION REDUCE A SECOND COMPOUND. WHAT?
    NITRO BLUE TETRAZOLIUM (NBT) WHICH FORMS A PUPRPLE PRECIPITATE.

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