Soil Microbial Ecology - Soil Molecular Ecology Laboratory

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Page 64 CONCENTRATING THE DNA Your final volume will be 50µl. If this is too dilute for your purposes, add 2µl of 5M NaCl and mix. Add 100µl of 100% cold ethanol and mix. Centrifuge at 10,000-x g for 5 min Decant all liquid. Dry residual ethanol in speed vacuum desiccators, or air dry. Resuspend precipitated DNA in desired volume. DNA FLOATS OUT OF WELL WHEN LOADED ON A GEL You may have inadvertently transferred some residual Solution S4 into the final sample. Prevent this by being careful in step 17 not to transfer liquid onto the bottom of the spin filter basket. Ethanol precipitation is the best way to remove Solution S4 residue. (See concentrating DNA above) STORING DNA DNA is eluted in Solution S5 (10mM Tris) and must be stored at -20°C or it may degrade over time. DNA can be eluted in TE but the EDTA may inhibit reactions such as PCR and automated sequencing. CELLS ARE DIFFICULT TO LYSE If cells are difficult to lyse, 10-min incubation at 70°C, after adding Solution S1, can be performed. Follow by continuing with protocol step 5. REFERENCES: Procedure with modification from MO BIO Laboratories Inc (http://www.mobio.com)
Al-Agely and Ogram © 2004 Page 65
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SOS 43 SOS4303C / SOS5305C Soil Mic
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Page 3 LABORATORY SCHEDULE 08/29 Fi
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Page 5 notebook. The instructor may
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Page 7 3. AUTOCLAVING Autoclaving (
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Page 9 Glomaceae Acaulosporaceae Gi
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Page 11 3. POT CULTURE AM mycorrhiz
Page 13 and 14: Page 13 hold a certain volume of so
Page 15 and 16: Page 15 METHOD This will be a demon
Page 17 and 18: Page 17 E X E R C I S E 3 SOIL MICR
Page 19 and 20: Page 19 Fungi constitute another ex
Page 21 and 22: Page 21 6. Continue this procedure
Page 23 and 24: Page 23 QUESTIONS: 1. Discuss the u
Page 25 and 26: Page 25 OBJECTIVE: Train to use saf
Page 27 and 28: Page 27 MICROSCOPE OPERATION Micros
Page 29 and 30: Page 29 METHOD 1. Place a small dro
Page 31 and 32: Page 31 GRAM-NEGATIVE AEROBIC RODS
Page 33 and 34: Page 33 2. OBSERVE SOIL FUNGI Exami
Page 35 and 36: Page 35 arthrospores or chlamydospo
Page 37 and 38: Page 37 SERIES ANNELLOSPORAE: First
Page 39 and 40: Page 39 6. Mount smears immediately
Page 41 and 42: Page 41 E X E R C I S E 6 SOIL MICR
Page 43 and 44: Page 43 METHOD: EXAMINATION OF ROOT
Page 45 and 46: Page 45 E X E R C I S E 7 SOIL ALGA
Page 47 and 48: Page 47 The most probable number (M
Page 49 and 50: Page 49 MATERIALS: Soil samples (ag
Page 51 and 52: Page 51 E X E R C I S E 8 SOIL META
Page 53 and 54: Page 53 METHOD: RESPONSE TO CARBON
Page 55 and 56: Page 55 A BIOMETER VESSEL A G F B C
Page 57 and 58: Page 57 Further example of calculat
Page 59 and 60: Page 59 PROTOCOL • To the 2ml Bea
Page 61 and 62: Page 61 Mechanical lysis is introdu
Page 63: Page 63 HINTS AND TROUBLESHOOTING G
Page 67 and 68: Page 67 E X E R C I S E 10 SOIL RHI
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