Ludox Extraction Trial 2:

Procedure:

Steps 1-6 were carried out as written above.  Steps 7-11 will be done next Thursday.

(left) an image of the 50 ml tube after the 4300 x g 15 minute centrifugation.

(right) an image of the two 2 ml microfuge tubes after step 6.

These are the basic procedures we use.  However, we made some changes to them.  The first difference is that we sifted our soil sample before adding water.  This was done to minimize the ammount of large particles such as rocks and grass.  Next, used 2.7 ml of liquid from the soil sample. Additionally, we added 0.3 ml of 2.5% Glutaraldehyde to the soil liquid before injecting it into the Ludox.  This was done in order to try and prevent the cells in sample from disintegration during any of the following procedures, namely the centrifuagtion in Ludox.  Steps 3 and 4 were carried out as printed.  The remainder of the steps have yet to be completed.

The soil sample used was drawn from a mixture of 10 grams of soil and 20 ml of D.I. water.  The mixture was placed on a vortex mixer for 5 minutes before the sample was taken.  We allowed about one minute for the soil to settle before we drew our sample from near the top.

What organisms make up healthy soil?

DNA Barcoding – one single species

Meta Barcoding – all species in a category

PCR was inspired by thermophile bacteria

Process:

One Cell found after the first round of centrifugation:

Group 1

• Challenges
  • data varies greatly
  • time and cost constraints
  • used antibiotic surrogates to examine ecological health
• Application
  • MOTU: molecular operational taxonomic unit
  • APDP: amplicon pyrosequencing denoising program
  • dbRDA
  • TITAN: threshold indicator taxa analysis

Group 2:

• Purpose
  • method of metaborcoding used to morphology
• Challenges
  • distinguishing active and inactive cells
  • interpretation of HTS
• Application
  • extracellular DNA
  • distinguishing living and dead cells

Group 3

• Purpose
  • distribution through habitat types
• Challenges
  • comparisons limited to a low sample number
  • lack of standardization between studies
• Application
  • distribution depends on habitat

Group 4

• Abstract: geographic separation in two distinct lineages
• Purpose
  • geographic separation causes genetic variation
• chellenges
  • hard to genetically characterize organisms
• application
  • cox – 1 gene might work four our purposes

Group 6

• Purpose
  • effects of soil and vegitation on protists
• Challeneges
  • lack of protist specific primers
  • most studies focus on DNA and not rRNA
• Application
  • separately analyze ribosomal RNA and DNA

Group 7

• Purpose
  • realize large diversity of soil parasites protists

Group 8

• Purpose
  • wants to make universal Eukaryotic taxonomy database
• challenges
  • no databases at the moment
• Application
  • we could use UniEuk in our study

Isopycnic Centrifugation: Uses tiny glass particles to change the density of the centrifuge substrate

Metabarcoding: the combination of DNA taxonomy and high-throughput sequencing, as a tool for the rapid assessment and monitoring of biodiversity in mixed, bulk samples

How To Clasify

• Microscopic
  • difficult to isolate
  • many are morphological similar
• Molecular
  • genomic diversity is unknown
  • standard methods are not established

3 Ways to Extract DNA

• Single Isolate ID
• Metabarcoding
• Metagenomics