Sequencing the bacteria

Altogether there were 31 bacteria found in the Onehunga Lagoon.  They were selected for genetic sequencing, as this is the only way to identify them absolutely.  There were 14 from collection site 1, 9 from collection site 2 and 8 from site 3.  Of these 8 were bacteria that had been found in the mud collected at the water/mud interface and 23 from sea water.  However, this project was not a quantitative study and no conclusions can be drawn from the numbers at each location; for instance, some were discarded because they were too similar to another sample from a different location.

There were 43 samples in all to sequence: my water microbes, fungi from Chris and quarantined (Q) cultures for the ICMP from Rose.  The Q cultures are held in the Newhook Laboratory under very strict conditions, laid out in their permit.  Q cultures can not be moved from their containment facility, unless they are first de-natured, and no longer viable.  To do this, Park had to temporarily relocate one of the thermal cyclers, and incubate the Q cultures at 95oC for ten minutes.  Then it was safe for them to leave the Newhook Laboratory for the Genetics Laboratory to complete the DNA sequencing.

Park adds an extracting solution; it is very alkaline which helps break down the cell walls.

Park keys in an individual cycle of temperatures for specific samples.

Park adds different primers for bacteria and fungi.

With Park's guidance, I made and poured the running gel for the electrophoresis unit.  Once the gel was poured, the spacer racks were inserted at even distances apart.  When the gel set, the spacer racks were carefully removed, and samples were first coloured with dye, and then added to each space,

When the electrophoresis unit is turned on, the negatively charged DNA will be attracted to the positive charge.  Over time, this will form characteristic bands or ladders, like the marker chart below.

Here is a print out of the ladders for these samples.  It gives a visual representation (or marker) of the DNA present in each sample.  The plate with the DNA ladders is then placed in the molecular imager.  The resulting ladder map is read to ascertain whether the samples are sufficiently robust for the final stages of the sequencing process.