DNA EXTRACTION

DNA EXTRACTION (QIAFILTER Plasmid Midi Kits)

Hey everyone!

As you guys know, I'm currently working as a research assistant, helping my supervisor with her project. I can't share too much about it, but let's just say it's something related to vaccines. And trust me, the budget for this project is huge! 😏

Last week, I was tasked with performing DNA extraction from bacterial cultures. We used a commercial kit for this, and let me tell you, it’s expensive! So, I had to be extra careful to make sure everything went smoothly and avoid any costly mistakes. I'll be more than happy if this post can give you some insight into the process. 😉

Now, onto the procedure! I'll try to simplify it as much as I can, but if you're looking for a more detailed version,click here Oh, and fun fact—we didn’t have the specialized rack for this kit, so we had to get a little creative to make things work.

1. Preparing the materials and equiment needed,

Materials; 

• Bacterial cultures
• QIAfilter Plasmid Midi kits
• Waste tube (50 mL)
• Eppendorf tube x 4

Equiment; 

• Spectrophotometer
• centrifuge
• Serological pipette 5 mL x 8
• Serological pipette 10 mL x 2
• Handgun pipette

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DNA Extraction Procedure

1. Check the OD (Optical Density) of the overnight bacterial cultures. Ensure it’s not over 0.6. If it exceeds, dilute and incubate for another 30 minutes.

   • Why? OD measurement helps determine bacterial growth; too high means overgrowth, which can affect DNA yield and quality.

2. Harvest bacterial cells by centrifugation at 6000 x g for 15 minutes at 4°C.

   • Why? This step collects the bacterial cells by spinning down the culture, making it easier to extract DNA.

3. Resuspend the bacterial pellet in 4 mL Buffer P1 (containing RNase A and LyseBlue).

• Why? RNase A degrades unwanted RNA, and LyseBlue acts as a visual indicator to monitor cell lysis.

                                                         
Picture : Buffer P1 that has been added with RNase A and lyseblue

1. Add 4 mL Buffer P2, mix by inverting 4-6 times, and incubate at room temperature for 5 minutes.

   • Why? Buffer P2 contains alkaline agents that lyse the bacterial cells and release DNA.

2. Add chilled 4 mL Buffer P3, mix immediately by inverting 4-6 times, and proceed to the next step.

   • Why? Buffer P3 neutralizes the solution, precipitating unwanted cell debris and proteins.

3. Pour the lysate into the QIAfilter cartridge barrel and incubate for 10 minutes at room temperature.

   • Tip: Do not agitate; let the precipitate float to the top naturally.

4. Equilibrate the QIAGEN-tip 100 by applying 4 mL Buffer QBT and allowing it to empty by gravity flow.

   • Why? This step prepares the column for optimal DNA binding.

                                                      

Picture 2 : Buffer QBT flow through QIAGEN-tip 100

1. Filter the lysate into the equilibrated QIAGEN-tip by gently inserting the plunger.

   • Tip: Be gentle to avoid disrupting the filtration process.

2. Let the cleared lysate enter the resin by gravity flow.

                                         

Picture 3 : cleared filter lysate enter resin through gravity flow

1. Wash the QIAGEN-tip with 2 x 10 mL Buffer QC.

   • Why? Washing removes any impurities while retaining the DNA on the resin.

2. Elute DNA with 5 mL Buffer QF and collect it in a 50 mL tube.

3. Precipitate DNA by adding 3.5 mL isopropanol, mix well, and centrifuge immediately at >15,000 x g for 30 minutes at 4°C.

   • Why? Isopropanol helps in concentrating the DNA by making it insoluble in solution.

4. Wash the DNA pellet with 2-3 mL of 70% ethanol, then centrifuge at >15,000 x g for 10 minutes.

   • Tip: Be careful not to disturb the pellet when decanting the supernatant.

5. Air-dry the pellet for 5-10 minutes and resuspend in 50 µL of nuclease-free water.

   • Why? Air-drying prevents ethanol contamination, ensuring better downstream applications.

6. Determine DNA concentration using a Denovix spectrophotometer.

   • Tip: Ensure the sample is well-mixed before taking readings for accurate results.

                                               

Picture 4 : DNA concentration reading using Denovix 

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And there you have it! 🎉

It might seem a little complicated at first, but trust me, once you get the hang of it, it’s actually pretty fun. Hope this breakdown helps, and feel free to reach out if you have any questions!.