Holmes, Dawn 1-1
Oxalate Treating FeGel Samples for Genomic DNA Extraction
1. Reduce the gassing station and light a Bunsen burner to create a sterile environment.
2. While keeping the culture aerobic and sterilely remove the needed amount of culture
from the tube or bottle. Transfer to a sterile 15mL or 50mL tube.
3. Add an equal volume of TPE solution in order to dilute the culture without
significantly changing the osmolarity. Use the TPE solution with 100mM
NaPhosphate for fresh water cultures and the TPE solution with 300mM NaPhosphate
for marine cultures.
4. Add filter-sterilized oxalate in small amounts until the culture turns yellow.
Be sure to mix the sample between each addition of oxalate, so that all the iron is
solubilized evenly and the color can be seen clearly.
5. Centrifuge the oxalate treated culture for 20 minutes at 4oC and 4000 rpms.
6. Wearing gloves carefully pour off supernatant by slowly inverting the tube once. Be
sure to not mix the pellet into the supernatant by inverting partly and repeating.
7. Store the tubes on ice until gDNA extraction. These cannot be frozen or kept on ice
for more then one to two hours. The longer they are left on ice the more cells will
lyse, decreasing the amount and quality of gDNA you will be able to extract.
Fresh Water TPE
100mM Tris pH 7.0
10mM EDTA
100mM Sodium Phosphate
Marine TPE
100mM Tris pH 7.0
10mM EDTA
300mM Sodium Phosphate
Oxalate Solution (1L)
Ammonium Oxalate 28g
15g
Oxalic Acid