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.
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
No expensive equipment required.
Very cheap.
Competent cells are made from an overnight culture rather than a culture in log phase growth.
Transformation and recovery are performed in a single tube.
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 powder
amount
units
ddH 2 O
196.7
mL
MS Powder (MP)
0.443
g
0.5 M MES (pH 5.7)
0.8
mL
plant agar or agarose (to get 0.5%)
1
g
add after autoclaving:
40% sucrose
2.5
mL
desired volume
200
mL
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
Notes:
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
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.
Gel recipe: 25 mL 1x TAE buffer, 0.3 g agarose –> boil by microwave until dissolved (about 45-55 sec on our microwave) —> add 1.25 µL 10 mg/mL ethidium bromide
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.
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
Large yield
High purity
Fast
Cheap, 20 times less than most commercial kits.
Tips
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.
This video shows you how to determine the molecular weight (MW) of a protein that was run on an SDS-PAGE gel. Note that the exact same technique can be used for determining the size of DNA fragments on agarose gels.
