bme molecular biology experiment - wordpress.com€¦ · 3/9/2017 · bme molecular biology...
TRANSCRIPT
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BME
MOLECULAR BIOLOGYEXPERIMENTMINIPREP & ENDONUCLEASE RXSKKU BME
3RD GRADE, 2ND SEMESTER
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TODAY
• Mini-preparation of plasmid DNA
• How to confirm whether the plasmid DNA is correct or not?
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MINIPREP
• Prep. the plasmid DNA from E. coli
• Minipreparation of plasmid DNA is a rapid, small-scale isolation of plasmid DNA from
bacteria. It is based on the alkaline lysis method. The extracted plasmid DNA resulting
from performing a miniprep is itself often called a "miniprep".
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WHAT’S THE FIRST STEP?
What’s this
^. ^
E. coli
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X_X
E. coli
WHAT’S THE FIRST STEP?
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LYSIS
• Traditional method
NaOH (0.2 N) and SDS (1%)
3 M NaoAC pH to 4.8 with glacial acetic acid
Lysis buffer
Neutralization buffer
What’s this
^. ^
E. coli
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LYSIS!
What’s inside?
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X_X
E. coli
HIGH PH
Neutralization for the next steps
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AFTER THE LYSIS...
X_X
E. coli
What is the main question with this situation after lysis?
What is your purpose?
Separate the plasmid DNA from others…
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AFTER THE LYSIS...
X_X
E. coli
Separate the plasmid DNA from others…
How?
1. Phenol-chloroform extraction
2. Using column
Idea> something capturing the plasmid DNA!!!
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PHENOL-CHLOROFORM EXTRACTION METHOD
Solution 1
Solution 3
Solution 2
Homework!
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COLUMN METHOD
DNA binding beads inside the column
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COLUMN METHOD
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DNA CONCENTRATION
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ONE MORE PROBLEM:LOW LOW CONCENTRATION!!!
• How to increase the concertation?
+
+
+
++
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ETHANOL PRECIPITATION
+
+
+
++
Consider the charge!
Hydrophilic and hydrophobic properties of plasmid DNA and ethanol
H.W.
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MIDIPREP
• Then what?
Maxiprep
Midipreparation
The starting E. coli culture volume is 15-25 mL
of lysogeny broth (LB) and the expected DNA yield is
100-350 µg.
Maxipreparation
The starting E. coli culture volume is 100-200 mL of
LB and the expected DNA yield is 500-850 µg.
Megapreparation
The starting E. coli culture volume is 500 mL – 2.5 L
of LB and the expected DNA yield is 1.5-2.5 mg.
Gigapreparation
The starting E. coli culture volume is 2.5-5 L of LB
and the expected DNA yield is 7.5–10 mg.
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HOW TO CONFIRM WHETHER THIS PLASMID IS CORRECT ONE OR NOT?
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HOW TO CONFIRM WHETHER THE PLASMID DNA IS CORRECT OR NOT?
Think! <hint> Plasmid DNA information
is DNA sequence…
1. PCR
2. Restriction enzyme cutting & size
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HOW TO CONFIRM WHETHER THE PLASMID DNA IS CORRECT OR NOT?
Think!
2. Restriction enzyme cutting & size
What’s your plan?
Using two enzyme sites
a. BamHI
b. XhoI
Then, check the plasmid size…using DNA electrophoresis
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WHAT DO YOU EXPECT TO SEE ON THE GEL?IMAGINE THE PLASMID DNA…
? ? ?
Write down in your result and discussion sessions.
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WHAT WE WILL DO TODAY…
• Miniprep using column method
• Plasmid confirmation by enzyme cutting
• DNA electrophoresis
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TODAY’S FINAL FIGURE
1kb
DNA
ladder
Uncut
plasmid
BamHI
cut
plasmid
XhoI
cut
plasmid
???
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HOMEWORK
• Please explain the mechanism of ethanol precipitation for DNA
• What is the Phenol-Chloroform extraction? Please explain the mechanism.
• How to measure the protein and cell concentrations? Please find the proper wavelengths
and mechanisms.
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NEXT WEEK
• DNA cleaning
• Restriction enzyme cutting