DNA time!

BIOL 4126 is about both classical and molecular methods. So far we have been involved in classical methods or phenotyping. We have been growing our strains on different media, with different NaCl concentrations and at different temperatures.



To do molecular work we need to have DNA to use for example in PCR of the 16S rRNA genes.
On Monday Dr. Rainey gave a talk about DNA extraction and the use of the MOBIO kit to extract DNA from bacterial cells. He talked about different ways to break open calls (enzymatic and physical) and ways to recover DNA (binding to a charged membrane like a filter or by precipitation). He went over the MOBIO kit protocol before we started to do it in the lab. Dr. Rainey uploaded the MOBIO protocol as a PDF to our AirSet page (which he claims we are not looking at often - not sure how he knows this?).



Anyways, on to DNA extraction....
Each group extracted DNA from 6 strains (Dr. Rainey told us which 6 strains to use from our 26 strains based on the fact that 16S rRNA sequences are already available for some of the strains). The steps in the extraction involve a lysis step which is achieved by bead beating the cells followed by the addition of various solution as well as incubation and centrifugation steps. The recovery of the DNA from the solution of the cell lysis is achieved by adding the lysate to a spin filter ("basket") - the DNA binds to the membrane in the spin filter and the lysate flows through on centrifugation. After all 700ul of the lysate has been passed through the spin filter we added a wash buffer which contains ethanol to the spin filter. This wash buffer cleans any of the salts and soluble cells components off the spin filter and the DNA stays bound to the membrane. The final step is to add 50ul of solution 5 (which might be water?) To the spin filter and this recovers the DNA from the membrane and it come through to the tube on centrifugation. This protocol takes a lot of time from start to finish and so we had to wait until Wed class to check our DNA.

DNA or no DNA...
At start of class on Wednesday Dr. Rainey explained how to visualize DNA and how to prepare and run an agarose gel.
He told us of the dangers of Ethidium Bromide and that we should be careful when using and wear gloves at all time. Also all waste associated with ethidium bromide, the gels, tips, gloves, paper towels should be disposed of in the EthBr waste container and not in the autoclave or normal trash (it is collected by campus safety and disposed of in a safe and environmentally friendly way).

To prepare the gel we made 100ml of 1X TBE buffer (from 10X stock). To this we added 1g of agarose. After mixing we micro waved the solution to dissolve and melt the agarose. After the solution had cooled to around 50C we added 1.5ul of EthBr solution and poured the gel into the gel rig. Then we placed the comb in the gel before it solidified.

The samples of our DNA from Monday were then prepared for loading on the gel. We took 2ul of our DNA and added it to 2ul of gel loading dye (contains bromo-phenol blue dye, glycerol and TBE buffer) - the 4ul was then loaded to the gel. Before loading we places 150 ml of 1X TBE in the gel rig and removed the comb (has 14 wells in it).

In addition we added 0.5ul of molecular marker (or ladder) III to 2ul of gel loading dye and loaded this in the well after our samples on the gel. This marker is used as a size standard on the gel to compare the size of our isolated DNA fragments. After loading the samples on the gel we connected it to the power supply and ran it on about 60V for 30 minutes or more. While the gel was running we looked at the soil serial dilution plates (more below).


To observe the DNA on the gel we removed it from the tank and took it to the common equipment room to view using the BioRad Gel Doc apparatus. We placed the gel inside the Gel Doc on the transilluminator and turned on the white light. On the computer we used the software to zoom in or out on the gel. We then turned off the white light and turned on the uv light on the transilluminator. The DNA bands on the gel which are stained with EthBr appeared as bright white bands on the image. The more DNA present the brighter the bands will be. There is a printer attached to the GelDoc which allowed us to print out our images - we also saved then as digital files and these are displayed below.


The gels of groups 1 and 2 ran on the same power supply and so the DNA ran less distance into the gel than that of group 3 which was run on an additional power supply. From the gel images it is clear that some groups obtained better DNA than others. The fact that some of the bands in the gel of group 3 are not visible does not mean the DNA is not present at concentrations that could be used in PCR. One of the students from Group 3 will redo the DNA extractions on their 6 strains. So we have DNA that can now be used in PCR during a future class.

Group 1 gel:


Group 2 gel:

Group 3 gel:



Gamma radiation kills many but not all bacteria....

We compared our serial dilution plates of the soils we plated on 1/21 and 1/26. There was a clear difference between the plates for the irradiated soil samples and the unirradiated samples. The numbers of colonies on the plates for the irradiated soils are a lot less than on the plates of the unirradiated soil samples.

There are many interesting observations on the effects of culture media and radiation. One interesting observation is for the Gobi soil sample - very few cells after irradiation form colonies on Marine Agar (which contains 3% salt) while there is extensive growth on Marine Agar for the Gobi soil that was not irradiated. There are also differences in the number of highly pigmented colonies between the culture media as well as between the irradiated and unirradiated soils. There is a lot can be written about and many comparisons that can be made between these plates. Dr. Rainey went over the plates with each group and discussed what the various observations and differences and why they might be.

We clearly have a lot of colonies on both sets of plates that could be representatives of the genera Blastococcus and Modestobacter. There are no black colonies as yet but then we have to consider that some Geodermatophilus species start out pink before turning green/brown/black as they age. Each student made observations of their plates and wrote these up in their lab book. Many will describe these observations in the blog above. We will count the colonies on these plates and work out the total cfus (colony forming units) per gram of soil before and after irradiation after they have been incubating for 20 days (no doubt the AirSet will send us a reminder....). After we counted the colonies we will pick colonies for further study and inclusion in our project. We plan to photograph the plates so as we can show the visual differences we have observed on our blog.

Finally we all registered at EZTAXON which we are going to use in class to analyze 16S rRNA gene sequences at a future date.