Hello all, I'm trying to use electrophoresis to separate 3 operons that are 500-600bp, for gel extraction and direct sequencing. When I check my PCR products on a 1% agarose TBE gel, I have clear bands. But when I run the same PCR product on a 1% agarose TAE gel, there are no bands. The DNA ladder shows up fine, positive controls are a go. The wells fluoresce, but because my expected product size is within the scope of the ladder it should not have run off the gel or remain in the wells. Streaks from the PCR primers show up on the gel, but no products. I'm running the TAE gel at 70 volts for 1.5 hrs (low and slow that is the tempo). This is my first time using TAE for gel extractions, am I doing something fundamentally wrong here? Any help is appreciated!
Hi Chelsea,
I am using TAE to prepare my gels and run them under similar conditions as you mention above and never experienced this kind of problems.
What do you use to stain your gel?
Have you purchased your TAE from a company or have you prepared it yourself? Could it be that there is something wrong with the buffer itself?
Hope you find a solution soon!
best regards,
Maria
Hello Chelsea,
I feel that you may have already solved the problem by now. But still in case....
This could be a random issue. We do extractions in TBE and many times TAE too. We have never had any issues till now. And we do make it ourself in the lab (just as you must be doing probably).
But I feel curious on two points:
1. Why to shift to TAE?
2. How many times has this occurred till now?
Prashant Singh I do not in fact have this problem solved quite yet. But I was of the understanding that you can't extract from TBE based gels because it interferes with sequencing reactions. Are you using a technique to prevent this? I made the buffers myself and as it was only my second time making TAE perhaps I did it incorrectly. Yet the I made another batch I had the same problem. Thanks for answers!
Hi Chelsea, TBE buffer isolated bands are amplified just fine. I tested that.
Hi Chelsea,
We do not do any modification with the TBE for sequencing reactions. But, its still very strange to read that TAE is not working. I'll suggest that you can use TBE (which is what you must be usually using).
Although you must have solved this issue by now, just out of curiosity, cross check that the Glacial Acetic acid is fine.
The difference in band visibility between a 1% TBE (Tris-Borate-EDTA) buffer gel and a 1% TAE (Tris-Acetate-EDTA) buffer gel could be due to several factors. Here are a few possibilities to consider:
To troubleshoot the issue, you could try the following:
- Verify the pH and prepare fresh TAE buffer.
- Double-check the gel concentration and ensure it is consistent for both gels.
- Use the same voltage and run time for both gels.
- Consider staining the gels with a different dye or visualization method to ensure consistent results.
By systematically addressing these factors, you may be able to identify the cause of the discrepancy in band visibility between the TBE and TAE gels.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
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