New Protocol for DNA Isolation from Bacteria

I used new kit to isolate my Agrobacteria's DNA during last week. New Kit has a simple and easy way to isolate pure DNA cultured bacteria. The kit contains all of the reagents needed to isolate and purify genomic DNA from gram-negative bacteria.

At first, I started to dilute Wash Solution Concentrate. I added 80 mL of ethanol 90%  to the Wash solution bottle which contained 10 mL of solution. The next step was preparing Proteinase K for the experiment. I dissolved Proteinase K powder in a bottle in 0.5 mL of Water RNA/DNA.

For the first step, I poured 1.5 ml of 18 hours old Agrobacteria into a centrifuge tube and centrifuged it at 12,000–16,000 × g for 2minuts. After centrifuging, I pipette out the culture medium completely. Then, I added 180 μL of Lysis Solution T and 20 μL of RNase A Solution in the tube. After 2 minutes incubating at room temperature, I Add 20 μL of the Proteinase K solution to the solution. Then, I placed tube into a 55 °C water bath for 30 minutes. After that, I added Add 200 μL of Lysis Solution C into the centrifuge tube and vortex it for 20 seconds to have a homogeneous mixture and  I put it back to the 55 °C water bath for 10 minutes. Then, I Add 500 μL of the Column Preparation Solution to each pre-assembled GenElute Miniprep Binding Column which is seated in a 2 mL collection tube and I Centrifuged it at 12,000 × g for 1 minute. The eluate was discarded. For the next step, I Added 200 μL of ethanol 90% to the solution and vortex it for 5–10 seconds to have a homogeneous mixture. I transferred my solution in the centrifuge tube into the binding column and centrifuged it for 1 minute at ≥ 6500 × g. Then, I placed column in a new collection tube. Then, I added 500 μL of Wash Solution 1 to the column and centrifuged tube at ≥ 6500 × g for 1 minute and I placed the column in a new 2 mL collection tube. After that, I added Add 500 μL of Wash Solution to the column and centrifuge for 3 minutes at 13000 × g. After centrifuging, I emptied the collection tube and put a column back on it. Then, I Centrifuged the column again for 1 minute at the same speed. The collection tube was discarded and column was placed in a new collection tube. At the end, I added 200 μL of the Elution Solution to the column and centrifuged for 1 Minute at 6600 × g. for getting better result of elution I repeated this step one more time.

I used this purified DNA to run a PCR and an agarose Gel.

First DNA extraction

  Second DNA extraction

In my first DNA extraction, I used isolated DNA to run an agarose gel and for the second DNA extraction, I used PCR of isolated DNA.