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BIOTEC LAB E1 C5

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  1. TRANSFORMATION SCHEME. NAME THE 4 STEPS
    • 1. PREINCUBATION
    • 2. INCUBATION
    • 3. HEAT SHOCK
    • 4. RECOVERY
  2. IN THE PREINCUBATION STAGE CELLS ARE SUSPENDEDE IN A SOLUTION OF --- AND INCUBATED AT ---C. WHY?
    • CATIONS
    • 0C
    • COMPLEX WITH NEGATIVE CHARGED PHOSPHATES OF LIPIDS IN CELL MEMBRANE. THE LOW TEMP CONGEALS THE MEMBRANE, STABLE DISTRIBUTION.
  3. WHAT HAPPENS IN THE INCUBATION PHASE OF THE TRANSFORMATION SCHEME?
    • DNA ADDED.
    • FURTHER INCUBATION AT 0C.
    • THE CATIONS ARE THOUGHT TO NEUTRALIZE NEGATIVELY CHARGED PHOSPHATES IN THE DNA AND THE CELL MEMBRANE.
  4. DESCRIBE THE HEAT SHOCK PHASE
    • CELL/DNA BRIEFLY INCUBATED AT 42C AND THEN RETURNED TO 0C.
    • CREATES A THERMAL INBALANCE ON EITHER SIDE OF E.COLI MEMBRANE-->PLASMIDS INTO CELL
  5. DESCRIBE THE RECOVERY STAGE OF THE TRANSFORMATION SCHEME.
    • LB BROTH IS ADDED TO THE DNA/CELL SUSPENSION AND INCUBATED AT 42C (IDEALLY WITH SHAKING) B4 PLATING ON SELECTIVE MEDIA.
    • TRANSFORMED CELL RECOVER FROM TREATMENT, AMPLIFY AND EXPRESS.
  6. DEFINE TRANSFORMATION EFFICIENCY
    EXPRESSED AS THE NUMBER OF ANTIBIOTIC-RESISTANT COLONIES PER MICROGRAM OF PLASMID DNA
  7. WHAT'S THE OBJECTIVE OF TRANSFORMATION EFFICIENCY?
    DETERMINE THE MASS OF pAMP THAT WAS SPREAD ON THE EXPERIEMNTAL PLATE AND THEREFORE RESPONSIBLE FOR TRANSFORMANTS OBSERVED.
  8. HOW DO YOU DETERMINE TOTAL MASS OF pAMP IN TRANSFORMATION?
    CONCENTRATION pAMP * VOL OF pAMP
  9. HOW TO DETERMINE THE FRACTION OF CELL SUSPENSION SPREAD ON A PLATE.
    • VOLUME SUSPENSION SPREAD /
    • TOTAL VOLUME SUSPENSION
  10. HOW TO EXPRESS TRANSFORMATION EFFICIENCY INSCIENTIFIC NOTATION AS THE NUMBER OF COLONIES PER ug OF pAMP.
    • NUMBER OF COLONIES OBSERVERED / MASS pAMP IN CELL SUSPENSION SPREAD.
    • (BASICALLY HOW MANY CELLS ONE GETS IN).
  11. T OR F?
    pAMP IS DERIVED FROM pUC EXPRESSION VECTOR THAT REPLAICATES TO A HIGH NUMBER OF COPIED CELLS.
    TRUE
  12. WHAT FACTORS MIGHT INFLUENCE TRANSFORMATION EFFICIENCY?
    • E. CLOI WITH LIGATED DNA. COMPOSED OF RELAXED CIRCULAR PLASMID AND LINEAR PLASMID DNA.
    • (GOOD TRANSFORMATION IS BETTER WITH PURIFIED, INTACT PLASMID DNA)
Author
shockwave
ID
104269
Card Set
BIOTEC LAB E1 C5
Description
BIOTEC LAB E1 C5
Updated
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