BME MOLECULAR BIOLOGY EXPERIMENT TRANSFORMATION & SPREADING · 9/1/2017 · BME MOLECULAR BIOLOGY...
Transcript of BME MOLECULAR BIOLOGY EXPERIMENT TRANSFORMATION & SPREADING · 9/1/2017 · BME MOLECULAR BIOLOGY...
BME
MOLECULAR BIOLOGYEXPERIMENT TRANSFORMATION & SPREADINGSKKU BME
3RD GRADE, 2ND SEMESTER
PIPETTE MAN
• Caution!
• How much can you push?
• Upside down?
• Suck more and more?
BROTH FOR BACTERIA AND VIRUS
BROTH FOR MICROORGANISM
LYSOGENY BROTH
• 10 g tryptone
• 5 g yeast extract
• 10 g NaCl
Why???
HOW CAN YOU SELECT ONE TYPE OF E. COLI?
TWO WAYS
• Antibiotics
• Colony
THEN HOW??
Antibiotics-resistance
X X X XX XX XX X
PENICILLIN
Alexander Fleming
ANTIBIOTICS
• Ampicillin & Penicillin
Β-LACTAM ANTIBIOTICS
• β-lactam antibiotics (beta-lactam antibiotics) are a class of broad-spectrum antibiotics, consisting of all antibiotic agents that contain a beta-lactam ring in their molecular structures.
WHY DO YOU KNOW THE MECHANISMS OF CHEMICALS AND PROTEINS IN MOLECULAR BIOLOGY EXPERIMENT?
• You can see as much as you know.
• For making a breakthrough
• For trouble shooting
ANTIBIOTICS MECHANISM
THEN, WHAT SHOULD YOU DO?
• LB
• Antibiotics
• Then?Live or die?
THEN, WHAT SHOULD YOU DO?
• LB
• Antibiotics
• Then?Something for antibotics-resistance
NOW, WHAT DO WE WANT TO DO?INSERT THE DNA INTO THE E. COLI!!!
• Transformation + 2 more ways (H.W.)
TRANSFORMATION
OK! You can use this transformation in lab.
Basic key question:Why is this transformation necessary for E. coli?
TRANSFORMATION
How to insert the plasmid into the cell?
Hint>What’s the barrier of E. coli?How to make it permeable?
TWO WAYS OF TRANSFORMATION
IMAGINE
THINK THE PROBLEMS!!!
AFTER HEAT SHOCK TRANSFORMATION
• Seed this in the LB + antibiotics èWhat do you expect?
AFTER HEAT SHOCK TRANSFORMATION
• Seed this in the LB + antibiotics èWhat do you expect?
AFTER HEAT SHOCK TRANSFORMATION
• Seed this in the LB + antibiotics èWhat do you expect?
• Culture without antibiotics for a while…
NEXT PROBLEM – CONTAMINATION?OR NON-WANTED PLASMID?
• How to select one?
One plasmid in one E. coli group!!!
HOW TO SEPARATE EACH PLASMID IN E.COLI?
• One plasmid in one E. coli
• Then?
• Low concentration culture?
• How to pick up one plasmid-transformed E. coli?
THINK YOU SPREAD LOW CONCENTRATION OF E.COLI ON THE LA PLATE…
IMAGINE
START EXPERIMENTMaking broth
Transformation
Spreading E. coli
LET’S MAKE THE BROTH FIRST!
BROTH FOR MICROORGANISM
• Lysogeny broth (LB)
• Lysogeny + Ampicillin
• Lysogeny + Kanamycin…
• Selection broths
PREPARATION FOR THE BROTH
• 10 g tryptone
• 5 g yeast extract
• 10 g NaCl
• Clean water
• Mass cylinder
• Flask
• Petri dish
• Autoclave
• Aluminum foil
• Autoclave tape
PREPARATION FOR TRANSFORMATION!
• Competent cell
• Plasmid DNA
• Selection marker
• Transformation media
• Heat block
• Water bath
PREPARATION FOR SPREADING
• Transformed cell
• LB + antibiotics agar plate
• Spreader
CLEAN BENCH
• BUT
• You can use magic spells!!
CLEAN!!!!
PREPARATION FOR SPREADING
• Transformed cell
• LB + antibiotics agar plate
• Spreader
• Alcohol lamps
HOW ABOUT VIRUS?
Max D.
HOMEWORK
• Why are the tryptone, yeast extract and NaCl necessary for LB?
• How does microorganism get the antibiotics-resistance? Explain the gene and mechanism.
• Find three ways of gene transfer in E. coli.
• Please find the ampicillin-resistance gene and explain its mechanism.
• Who is Max Delbruck (virus counting method)?
• How to culture the influenza virus? Which broth, media and host are necessary?
• What are the critical factors to make a primer for PCR?
• What is the annealing temperature in PCR? How to calculate it?
NEXT WEEK
• PCR of template
• Vector seeding