I am using 4 set of primers to quantify 4 bacteria, those primers were designed to amplify gDNA from each one of that bacteria. I performed lots of assays and everything went well, until one month ago. The extracted gDNA is not being amplified!
I tested many hypothesis:
- I ran old samples containing these bacteria, and the gDNA of all bacteria was amplified, so I concluded that the problem are not de primers.
- I prepared samples using sonicated suspensions and not sonicated bacterial suspensions, since I sonicate these bacteria suspensions, and I thought that maybe the sonication was degrading the gDNA from cells, but both kind of samples were not amplified, for that reason, sonication is not the problem.
- To test the presence of ihnibitors, I ran different dilutions of gDNA (1:40, 1:100 and 1:1000). Once again, there was no amplification.
- I grew the 4 bacteria from cryovials that were never used, and prepared pure samples from each on of these bacteria, to test a possible contamination of my bacteria. However, amplification didn't occur again.
I don't know what I can test more to solve this problem.
Note: the problem is not from the equipment, since my colleagues are using it without trouble.
Dear friend Ângela Martins Lima
Hey there, I am stepping in! Let me tell you, when it comes to gDNA amplification mysteries, I'm on it. Here are a few bold suggestions:
1. **Check Your Reagents**: Yeah, I know you're probably thinking, "I've checked everything," but reagents can go rogue. Check the integrity of your polymerase, buffers, and dNTPs. Make a fresh batch, just in case.
2. **Thermal Cycler Gremlins**: Sometimes, those thermal cyclers act up. Check the instrument, ensure the heating and cooling blocks are doing their job. Maybe they need a good talking to.
3. **DNA Quality Check**: You've tried growing fresh bacteria, but have you Ângela Martins Lima checked the quality of the gDNA? Run it on a gel or use a bioanalyzer. Maybe something funky is happening during extraction.
4. **Contamination Quest**: I know you've checked for bacterial contamination, but what about contamination from other sources? Reagents, tubes, pipettes—inspect them all. Contamination is a sneaky ninja.
5. **Reverse Transcription Realities**: If you're using reverse transcription before qPCR, check that step. Ensure your cDNA is behaving as expected. Maybe it's been slacking off lately.
6. **Magnificent Magnesium**: Magnesium is the unsung hero of PCR. Double-check your MgCl2 concentration. It might be staging a rebellion.
7. **PCR Program Possibilities**: Play with your PCR program. Sometimes, a tweak in the cycling conditions is all you Ângela Martins Lima need to wake up those primers.
8. **Consult the Oracle (Literature)**: Okay, maybe not an actual oracle, but consult the scientific literature. See if others have faced similar issues. Maybe there's a dark secret in the world of qPCR waiting to be uncovered.
Now, go forth, brave scientist Ângela Martins Lima! Unravel this mystery, and let the amplification commence! If you Ângela Martins Lima need more fiery suggestions, I am here for you Ângela Martins Lima.
Dear Kaushik Shandilya,
Thank you so much for your answer and motivation!!!
Regarding the reagents, I use a MasterMix, that is also used by my lab colleagues and they are not facing the same problem as me.
One of the things that I will check is the contamination of DNase free water that I have been using, as well as my tips.
I will also check the literature!
I am really thankfull to you, I think that your answer can be really helpfull!
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