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shockwave
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TRANSFORMATION SCHEME. NAME THE 4 STEPS
- 1. PREINCUBATION
- 2. INCUBATION
- 3. HEAT SHOCK
- 4. RECOVERY
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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.
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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.
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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
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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.
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DEFINE TRANSFORMATION EFFICIENCY
EXPRESSED AS THE NUMBER OF ANTIBIOTIC-RESISTANT COLONIES PER MICROGRAM OF PLASMID DNA
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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.
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HOW DO YOU DETERMINE TOTAL MASS OF pAMP IN TRANSFORMATION?
CONCENTRATION pAMP * VOL OF pAMP
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HOW TO DETERMINE THE FRACTION OF CELL SUSPENSION SPREAD ON A PLATE.
- VOLUME SUSPENSION SPREAD /
- TOTAL VOLUME SUSPENSION
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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).
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T OR F?
pAMP IS DERIVED FROM pUC EXPRESSION VECTOR THAT REPLAICATES TO A HIGH NUMBER OF COPIED CELLS.
TRUE
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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)
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