Why do my PCR products look like this on my gel? The products are amplified using LAMP specific for E. coli. Lane 1 contains a 1kb ladder, 2 contains E. coli and 7 contains a negative control. Lanes 3, 4, 5 and 6 contain bacteria that are not E. coli. I used a 1,2% agarose gel and 1X TBE buffer.
Hi
The problem might be with the running buffer. The DNA makers is also not showing an expected pattern at all.
Could the buffer be too concentrated? We have always used a 1X TBE buffer in previous gels and have never obtained results like this.
It may be that your gel comb is too deep into the agarose and the bottom of the well is splitting when you remove the comb allowing sample mixing while loading and the sample is running under the gel not through it. Try cleaning the comb well and putting a spacer under it to lift the teeth higher into the gel and look for samples spreading before the electrophoresis is started
Hi
As mentioned by Paul Rutland above leakage seems to be possible in the later samples as even dye is also not visible. I agree 1XTBE is also what we use on a regular basis. However since even DNA ladder is also not visible, you may need to re-run and see if only the ladder is well resolved or not, if not buffer concentration/pH may be the culprit.
Hi Ella Peeters
The problem with your gel may be of several reasons:
1. Voltage fluctuations when running the gel - Main reason
2. Improper preparation of agarose gel (improper melting and casting of agarose will lead to ununiform concentration of agarose throughout the gel).
3. Problem with your PCR machine (Heating block may not be working properly)
4. Problem with your PCR reaction preparation such as DNAse contamination, old chemicals, forget to add any of the components, contains PCR inhibitors (ethanol not removed properly at last step of DNA isolation), wrong PCR condition, and so on.
So, take time to perform each step starting DNA isolation, PCR setup, and gel run.
I hope my answer will be helpful...
Thank you and All the Best...
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