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The Molecular Biology Labs - LAB 4
LISTS | LAB 1 | LAB 2 | LAB 3 | LAB 5 | LAB 6 | LAB 7 | LAB 8 | LAB 9 | APPEND 1 | APPEND 2

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The Molecular Biology Labs


LAB 4: Estimation of DNA Concentration

| MATERIALS | SUPPLIES | PROCEDURES | RESULTS |

Materials:

  1. TE solution
    • 10 mM Tris (pH to 7.5)
    • 1 mM EDTA (pH to 8.0 to dissolve)
  2. DNA sample
  3. SYBR Green I(R) nucleic acid gel stain (Molecular Probes)
  4. Plastic wrap
  5. distilled water
  6. DNA marker stock (10 mg/ml)
Supplies:
  1. Tubes
  2. Polaroid setup (with proper filter - SYBR Green/Gold gel stain photographic filter) and UV light box Teacher's note: UV is dangerous to the skin and eyes, so supervision is very important.
  3. Micropipetter and tips
 Procedures:
  1. Prepare 6 DNA standards from DNA marker stock:
    • standard I (5 ug/ul): 1:2 dilution of DNA marker stock
    • standard II (2.5 ug/ul): 1:2 dilution of standard I
    • standard III (1.25 ug/ul): 1:2 dilution of standard II
    • standard IV (0.625 ug/ul): 1:2 dilution of standard III
    • standard V (0.313 ug/ul): 1:2 dilution of standard IV
    • standard VI (0.156 ug/ul): 1:2 dilution of standard V
  2. Make a 1:5000 dilution of the SYBR Green I(R) with TE solution.
  3. Mix 5 ul of the DNA sample and each of the 6 standards with 5 ul of the diluted SYBR Green I(R) dye.
  4. Place a sheet of plastic wrap smoothly onto the UV light box.
  5. Spot the mixtures individually onto plastic wrap.
  6. Spot the set of 6 standards.
  7. Turn on the UV light and take a photo (Polaroid 667 black-and-white print film). Teacher's note: UV is dangerous to the skin and eyes, so supervision is very important.
  8. Compare the brightness of the DNA sample with the DNA standards and estimate concentration.
 Results:
  1. If concentration of DNA sample appears to be outside of the range covered by the standards, repeat with a different set of standards.

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