report draw a scheme of the ga20ox cloning procedure
TRANSCRIPT
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Report
• Draw a scheme of the GA20OX cloning procedure.
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Question 1: Why do we study the effect of GA on alapha-amylase in embryoless seeds and not on whole seeds?
Question 2: Volumes
Question 3: Why do we do treatment 4? What is the difference to treatment 3?
Question 4: What is the "debris"?
Question 5: Why is the decrease in absorbance proportional to the quantity of alpha-amylase in the reaction mixture?
Question 6: What do you do if your absorbance in your extract is out of the range of your standard curve?
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Western Blot
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Steps:
1. SDS-PAGE2. Transfer to membrane: "Blotting"3. Detection of proteins
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SDS-PAGE
SDS: sodium dodecyl sulfate
PAGE: polyacrylamid electrophoresis
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The goal is to separate proteins according to their sizes.
How would you do that?
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Remember
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SDS: sodium dodecyl sulfate is a detergent (soap) that can dissolve hydrophobic molecules but also has a negative charge (sulfATE) attached to it
http://www.davidson.edu/academic/biology/courses/Molbio/SDSPAGE/SDSPAGE.html
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www.ufs.ac.za
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Reductant:
DTT: DithiothreitolB-Mercaptoethanol
The reducing agent beaks any cystine (-S-S-) bonds formed between two cysteine residues
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Other stuff in the sample buffer:
Glycerol
Bromphenolblue
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How to make the gel?
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Polymerization reaction: radical catalyzed reaction
http://www.davidson.edu/academic/biology/courses/Molbio/SDSPAGE/SDSPAGE.html
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Polymerization reaction
http://www.davidson.edu/academic/biology/courses/Molbio/SDSPAGE/SDSPAGE.html
Catalysts:
APS: Ammonium persulfate, radical initiator
TEMED:N, N, N', N'-tetramethylethylenediamine, free radical stabilizer
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"Discontinuous" PAGE
Low pH (6.8)Low ionic strengthLow Acrylamid concentrationFAST
High pH (8.8)High ionic strengthHigh Acrylamid concentrationSLOW
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Visualization of proteins on the gel: Coomasie stain
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OR do a western blot
Proteins are transferred to a protein binding membrane.
We will use a nitrocellulose membrane.
Polyvinylidene difluoride (PVDF) is also commonly used.
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OR do a western blot