I am a Year 4 student now working for my final year project. My topic is using cerium oxide nanoparticles system to remove the sul1 gene in RP4 E. coli. In my preliminary study, we discovered that the nanoparticle can completely degrade the gene sequence, so my supervisor suggest me to make a research based on this.
Here are my antibacterial system, which consists of 1.5mM CeO2 NPs, 0.004mM H2O2 and 2mM KBr, the principle of antibacterial is haloperoxidase-like activity. However, in the DNA extraction part I can only extract 0.003ng/ul DNA (the most)....In my process, I take out the RP4 E.coli from -80 degree refrigerator and take 1ml into 10ml LB broth and incubate for 6 hours, normally, the OD600=0.7~0.8. Then I will take 1ml of the bacterial suspension to centrifuge for 8000rpm, 10min, the pellet was small, not to mention the pellet in the antibacterial system, after centrifuge, there were only nanoparticles in the bottom (and some bacteria that I cannot see).
My stock solution is 15mM CeO2, 0.04mM H2O2 and 20mM KBr, I added 5ml each of this component and added 6ml of the bacterial suspension in a sterile conical flask. Then I put the conical flask into the shaking incubator 37degree 220rpm, then I took 1ml sample at 3min, 10min, 20min and 30min and put then on the ice to stop the reaction. After all the samples were ok, I followed the protocol of QIAamp DNA Blood Mini Kit to extract the DNA, originally in the quick start protocol the 56 degree waterbath only take 10 min, but I think the time is too short for the protease K and buffer AL to work, so I extend the time to 1 hour, then I will repeat the buffer AW2. However, the result from Nanodrop were very bad that all the DNA concentration were extremely low, and the ratio of A260/280 are much higher than 3 and the A260/230 are only around 0.1~0.3. Regardless of these bad result, I ran a real-time PCR, and it turns out that almost all the Ct value are around 30, there are no difference between 0min and 30min nano treatment.
What can I do? Please help me...I am running out of time.
From a 1ml culture of E. coli cells you should get lots of DNA. It might be that the Blood DNA kit does not work well on bacteria. I would use a kit or follow a protocol that lyses bacteria. You can find many protocols in the literature if you just do a search or there are lots of companies that sell DNA preparation kits that are optimized for bacteria.
Michael J. Benedik Thank you for your answer and yes I know that the DNA yield is strange, but in our university, we use this mini kit for quite a long time and in this prototol there are instructions for bacteria, it was no change even I follow the protocal for bacterial suspension in the detailed instruction......So I am very confused now
If the kit is known to work then either you are not starting with sufficient cells (are the cultures growing well?) or you are losing your sample along the way. Is there someone else in the lab that has done this procedure and who can watch or show you, it might just be some simple procedural error.
@Michael J. Benedik I am afraid no. Because I have more than one year experience of RA, and for these processes I have repeated many times and there was a professor watch me to extract the DNA with this kit last year and there was'n any problem...
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