DNA extraction from plants – recap Smush (“homogenize”) the cells Dissolve membranes and...
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Transcript of DNA extraction from plants – recap Smush (“homogenize”) the cells Dissolve membranes and...
![Page 1: DNA extraction from plants – recap Smush (“homogenize”) the cells Dissolve membranes and solublize DNA in a detergent buffer with nuclease inhibitors Remove.](https://reader035.fdocuments.net/reader035/viewer/2022062321/56649e765503460f94b776af/html5/thumbnails/1.jpg)
DNA extraction from plants – recap
• Smush (“homogenize”) the cells• Dissolve membranes and solublize DNA in
a detergent buffer with nuclease inhibitors• Remove solids with centrifugation• Remove proteins with KOAc precipitation• Precipitate DNA with alcohol• Remove DNA-bound proteins with phenol-
chloroform and chloroform extraction
![Page 2: DNA extraction from plants – recap Smush (“homogenize”) the cells Dissolve membranes and solublize DNA in a detergent buffer with nuclease inhibitors Remove.](https://reader035.fdocuments.net/reader035/viewer/2022062321/56649e765503460f94b776af/html5/thumbnails/2.jpg)
Waiter!
There’s RNA in my food too…
![Page 3: DNA extraction from plants – recap Smush (“homogenize”) the cells Dissolve membranes and solublize DNA in a detergent buffer with nuclease inhibitors Remove.](https://reader035.fdocuments.net/reader035/viewer/2022062321/56649e765503460f94b776af/html5/thumbnails/3.jpg)
DNA
A stable, double helix designed to store information and be chemically unreactive
![Page 4: DNA extraction from plants – recap Smush (“homogenize”) the cells Dissolve membranes and solublize DNA in a detergent buffer with nuclease inhibitors Remove.](https://reader035.fdocuments.net/reader035/viewer/2022062321/56649e765503460f94b776af/html5/thumbnails/4.jpg)
T. thermophilus - Ramakrishnan et al., Cell, 2002
RNA
A different tale
![Page 5: DNA extraction from plants – recap Smush (“homogenize”) the cells Dissolve membranes and solublize DNA in a detergent buffer with nuclease inhibitors Remove.](https://reader035.fdocuments.net/reader035/viewer/2022062321/56649e765503460f94b776af/html5/thumbnails/5.jpg)
![Page 6: DNA extraction from plants – recap Smush (“homogenize”) the cells Dissolve membranes and solublize DNA in a detergent buffer with nuclease inhibitors Remove.](https://reader035.fdocuments.net/reader035/viewer/2022062321/56649e765503460f94b776af/html5/thumbnails/6.jpg)
![Page 7: DNA extraction from plants – recap Smush (“homogenize”) the cells Dissolve membranes and solublize DNA in a detergent buffer with nuclease inhibitors Remove.](https://reader035.fdocuments.net/reader035/viewer/2022062321/56649e765503460f94b776af/html5/thumbnails/7.jpg)
![Page 8: DNA extraction from plants – recap Smush (“homogenize”) the cells Dissolve membranes and solublize DNA in a detergent buffer with nuclease inhibitors Remove.](https://reader035.fdocuments.net/reader035/viewer/2022062321/56649e765503460f94b776af/html5/thumbnails/8.jpg)
![Page 9: DNA extraction from plants – recap Smush (“homogenize”) the cells Dissolve membranes and solublize DNA in a detergent buffer with nuclease inhibitors Remove.](https://reader035.fdocuments.net/reader035/viewer/2022062321/56649e765503460f94b776af/html5/thumbnails/9.jpg)
![Page 10: DNA extraction from plants – recap Smush (“homogenize”) the cells Dissolve membranes and solublize DNA in a detergent buffer with nuclease inhibitors Remove.](https://reader035.fdocuments.net/reader035/viewer/2022062321/56649e765503460f94b776af/html5/thumbnails/10.jpg)
RNA extraction from plants
• Smush (“homogenize”) the cells
• Dissolve membranes and solublize RNA in a detergent buffer with RNAse inhibitors
• Remove solids with centrifugation
• Remove proteins with phenol extraction
• Remove DNA with LiCl precipitation
• Precipitate RNA with alcohol
![Page 11: DNA extraction from plants – recap Smush (“homogenize”) the cells Dissolve membranes and solublize DNA in a detergent buffer with nuclease inhibitors Remove.](https://reader035.fdocuments.net/reader035/viewer/2022062321/56649e765503460f94b776af/html5/thumbnails/11.jpg)
Chloroplast (rRNA, mRNA, tRNA)
Mitochondrion(rRNA, mRNA, tRNA)
Vacuole (RNAse!)
Cell Membrane
Cell Wall
Cytosol(mRNA, tRNA, rRNA)
Nucleus(DNA,snRNA,siRNAs)
![Page 12: DNA extraction from plants – recap Smush (“homogenize”) the cells Dissolve membranes and solublize DNA in a detergent buffer with nuclease inhibitors Remove.](https://reader035.fdocuments.net/reader035/viewer/2022062321/56649e765503460f94b776af/html5/thumbnails/12.jpg)
RNA extraction issues
• Homogenization • RNAse• DNA
contamination• pH of phenol
RNAse