Symbiotic Nitrogen Fixation

Soybean symbiosis with Rhizobia

Legumes (beans) are capable of symbiotic nitrogen fixation. The bacteria turn atmospheric nitrogen into ammonia that the plant can use. Soybean Rhizobia inoculum can really make soybeans grow much bigger and healthier without adding fertilizer. The time-lapse video below shows how beneficial Rhizobia can be to soybean growth. All pots had identical soil. The only difference is the seeds that went into the pots on the right had Cel-Tech soybean inoculum on them. The Rhizobia form a symbiotic relationship with the soybean plants leading to nodule formation on the roots. These root nodules provide the plants with an abundant source of nitrogen.

Soybean growth details

  1. Soil (Promix BX) was autoclaved to kill residual bacteria.
  2. Seeds were planted into wet soil. Plants with Rhizobia were made by touching the seeds to Cel-Tech Inoculant prior to planting.
  3. Plants were grown in 16 hours of light per day at 23 degrees Celsius. Plants were watered as needed.
  4. Note: Extreme care is needed to prevent non-inoculated plants from “catching” the Rhizobia from the inoculated plants (ie. do not cross contaminate by splashing water or soil between the treatments). Soybeans are very susceptible to Rhizobia colonization.

Here are several other plant time-lapse videos.

Statistical analysis, t-test in Excel

Statistical analysis

Statistics are one of the most important tools for a scientist. They allow scientists to test hypotheses. Perhaps you would like to  know if a treatment makes your favorite organism grow better. Statistical analysis would let you determine the probability that the treatment increases growth. The below video shows you how to perform statistical analysis (t-test) in Excel.

Choice of t-test (paired vs two sample equal variance vs two sample unequal variance)

Ideally statistical analysis should be planned before the experiment is setup. Paired t-test has more statistical power than the other two types of t-test because it helps minimize the effect of nuisance factors that confound the experiment results. An example of how to setup an experiment to take advantage of paired t-test in plants is as follows. Control and treatment for each replicate would be planted side by side so that the replicate was in a block. Doing this minimizes the effect that different growing positions have on each replicate. A paired t-test takes this into consideration and assumes while the absolute measurement values might change for different replicates, the mean difference between control and treatment for each replicate should be conserved. The best part of using a paired t-test or other blocking strategy, is that replicates do not need to be completed at the same time. This way there is no real limit to the number of replicates for an experiment.

Quantifying SDS-PAGE bands

Quantifying bands on SDS-PAGE using ImageJ

The below video shows you how to use ImageJ to quantify bands on a SDS-PAGE gel image. In this example, bands are normalized to the total protein in the lane.

Here you can download the sample gel image from the video to practice with. Note that many commercial gel docs, like Bio-rad, come with software for quantifying SDS-PAGE bands. In contrast, ImageJ is a free alternative that can work with any gel images including ones generated with an office scanner. Almost the same procedure in the video can be used for quantifying nucleic acid gels or even features on non-gel images.

Enzyme kinetics

Enzyme kinetics is the study of enzyme catalyzed chemical reactions. Enzymes speed up reactions by binding to the transition state of the reaction. A detailed overview on enzyme kinetics can be found here. Increasing substrate concentration speeds up a reaction to a certain point, Vmax. Vmax is the maximum reaction rate that can occur and adding more substrate will not increase the rate of the reaction because the enzyme binding site has become saturated. The Michaelis–Menten constant, Km, is defined as the substrate concentration in which the reaction will go at half maximal rate. In order to determine the Km and Vmax of a reaction you will first have to calculate the reaction rate at several different substrate concentrations.

Determining reaction rates

This video shows you how to calculate reaction rates in Excel for urease, an enzyme that catalyzes the hydrolysis of urea to carbon dioxide and ammonia.

Determining Km and Vmax

Km and Vmax can be determined once reactions rates at different substrate concentrations have been calculated. This video shows you how to calculate Km and Vmax in Excel.

Transforming Agrobacterium by the freeze thaw method

Plant transformation is frequently carried out with the aid of Agrobacterium. Agrobacterium is a naturally occurring bacteria that genetically modifies plants in the wild. If you have ever seen a tree with a large tumor on it, you have seen the work of Agrobacterium. Currently, scientists exploit the genetic engineering capabilities of a domesticated Agrobacterium to transform plants.

This video shows you how to transform Agrobacterium by the freeze-thaw method. Agrobacterium transformation by the freeze thaw method is cheap, fast and efficient. The transformation and recovery steps can be performed in a single tube. Making Agrobacterium competent cells for the freeze thaw method is easy because it is not necessary to capture the cells in log phase growth. Instead, an overnight culture of Agrobacterium can be used to make these competent cells. The freeze-thaw method does not require electroporation apparatus or expensive electroporation cuvettes. The freeze-thaw transformation method is not as efficient as electroporation, however, it always gives more than enough colonies. A written version of the Agrobacterium freeze thaw transformation can be downloaded here. Once you have your desired binary vector in Agrobacterium, you could easily transform Arabidopsis by Agrobacterium floral dip.

Advantages over electroporation

  1. No expensive equipment required.
  2. Very cheap.
  3. Competent cells are made from an overnight culture rather than a culture in log phase growth.
  4. Transformation and recovery are performed in a single tube.

Sterilizing seeds and selecting for hygromycin resistance

This video protocol shows you how to surface sterilize Arabidopsis seeds and select for hygromycin resistance. Hygromycin selection is one of the fastest and most clear cut selections for transgenic Arabidopsis. Unfortunately, hygromycin does not do a good job killing non-resistant plants. However, the selection outlined in this post is extremely fast and easy to do. The selection takes advantage of the fact that plants elongate in the dark and that hygromycin inhibits this elongation. An Arabidopsis Seed Sterilization Protocol for Screening can be downloaded here. Surface sterilized Arabidopsis seeds that have been cold treated overnight  should be put onto half strength MS plates with 50 µg/mL hygromycin for selection of transgenic plants. Plates are exposed to light for 4 hours followed by 5 days in the dark. Transgenic plants will have elongated hypocotyls. The recipe of 0.5x MS hygromycin is shown in the table below.

0.5x MS from MS powderamountunits
ddH 2 O196.7mL
MS Powder (MP)0.443g
0.5 M MES (pH 5.7)0.8mL
plant agar or agarose (to get 0.5%)1g
add after autoclaving:
40% sucrose2.5mL
desired volume200mL
add ingredients in order except sucrose solution to a flask
autoclave for 5-15 minutes on a liquid cycle
mix agar by swirling after autoclaving is finished
add indicated amount of sterile sucrose
if necessary, cool to 55-60 °C and add desired selection agent like glufosinate, kanamycin, or hygromycin
(ie. 200 µL 50 mg/mL hygromycin that was frozen at -20°C)
mix by swirling then pour plates
10x MS salts = 4.43 g MP Biomedicals (Cat. No. 2623122) per 100 mL, store at 4 °C
prepare sterile 40% sucrose by making 40 g sucrose per 100 mL H2O and autoclaving by itself
note: sucrose will break down partially if it is autoclaved with iron, which is a component of MS salts
note: plant agar is cheaper than agarose; do not use bacterial agar

You can also download an Excel sheet that can calculate the components for any volume of 0.5x MS plant agar or agarose from powder or stock MS salts here.

Using top agarose

Optionally you can plate your seeds using top agarose to get a very even spread and even clearer cut selection. This video illustrates the results with top agarose. Seeds were mixed with an equal volume of 70°C 0.5% agarose and 2 mL (1 mL of seeds) was plated.

Definitive and rapid selection of BASTA resistant plants on plates

The method on Jose Alonso’s website for selecting BASTA resistant Arabidopsis is excellent. In 5 days you can get a clear cut selection. The method involves plating seeds in top agar. Compared to all other plate based BASTA selections I have tried, this one is by far the best. Seeds are plated, cold treated, and light treated before putting them in the dark for 3 days. Then the plates are moved to the light for 2-5 days. Only BASTA resistant plants will develop a green cotyledon.

Transforming Arabidopsis by Agrobacterium floral dip

Transforming Arabidopsis is one of the easiest things to do. Easy transformation has certainly helped make Arabidopsis the premier model plant for scientific study. A printable protocol for Arabidopsis Transformation by Agrobacterium floral dip can be downloaded here. A cheap and efficient way to get your binary vector of choice into Agrobacterium is by the Agrobacterium freeze and thaw transformation method.


  • Other pages on on this website show how to select for transformants on sterile plates.
  • Healthy plants are really important for the transformation process to work.

Preparing and running slide gels

Preparing slide gels

Slide agarose gels can be run in 7 minutes and give great resolution of DNA samples. Multiple gels can be prepared at once and saved for up to a month. Slide gels use only 0.12 g of agarose per gel which reduces the cost of running gels. See the accompanying video “Running slide gels” below.

Gel recipe: 10 mL 1x TAE buffer, 0.12 g agarose –> boil by microwave until dissolved (about 45 sec on our microwave) —> add 0.5 µL 10 mg/mL ethidium bromide

Running slide gels

Slide agarose gels can be run in 7 minutes and give great resolution of DNA samples. Multiple gels can be prepared at once and saved for up to a month. Slide gels use only 0.12 g of agarose per gel which reduces the cost of running gels. See “Preparing slide gels” above for casting the gels.

Super cheap plasmid miniprep without columns

Short version (Recommended).
Long version with more tips and details but poor sound quality.

This video protocol shows you how to perform an alkaline lysis plasmid miniprep. The DNA purification industry would like you to believe that their kits are necessary to obtain plasmid DNA suitable for routine applications. This is simply not true. In fact, isolating large quantities of high purity plasmid DNA is really easy.  Plasmid miniprep procedures commonly use columns that give low yield and are relatively expensive. This modified version of the traditional alkaline lysis miniprep produces large yields of high quality DNA and costs less than $0.05 per prep (including cost of tubes). Download a printable plasmid miniprep protocol here. The protocol described here (and shown in the video) has a few modification from the classical miniprep protocol found in books such as “Molecular Cloning” that increase the purity of the plasmid DNA.

Advantages of this miniprep method

  1. Large yield
  2. High purity
  3. Fast
  4. Cheap, 20 times less than most commercial kits.


  • The hole in the cap of the tube can be taped shut after cultures have finished growing, when using 2 mL microcentrifuge tubes to grow cultures. Pelleted E. coli can be frozen at -20 degrees C after the supernatant is discarded and processed later.
  • It is important not to transfer and white precipitate to the new tube at step 8 in the protocol. However, you will have a second chance to get rid of the white precipitate later. When resuspending the ethanol pellet in Tris buffer (step 13) you may have some white precipitate that does not resuspend. This material is not DNA.  Therefore, you can spin the insoluble material to the bottom of the tube and transfer the clear liquid to a new tube.