Not only you get bands in the negative control but also get multiple bands in each lane. I'd try to design better primers and optimize the PCR program to get only one band and change all the reagents to avoid the contamination (it is also a good idea to do everything in another lab, if you have that possibility).

If you have a positive control for the PCR, include it so you'll be sure which band is the correct one.

To interpret the results of your gel electrophoresis, we need to consider several factors: the presence or absence of bands in each well, the size of these bands, and their comparison to the negative controls and DNA markers. Here's a step-by-step guide for interpreting your results based on the provided information:

1. Analyze the Negative Controls

• Wells 9 and 18 (Negative Controls): These wells should not contain any bands. The presence of bands indicates contamination in your PCR setup. If you see bands in these wells, it suggests that either your reagents, pipette tips, or environment are contaminated with the target DNA/RNA.

2. Analyze the Experimental Wells

• Experimental Wells (1-8 and 10-17): These wells contain the cDNA samples amplified by PCR. You should see distinct bands if your target genes (p53 and K-RAS) are present in the samples.Band Size: Compare the bands in these wells to the DNA markers (last two wells) to determine their size. The bands should correspond to the expected size of your p53 and K-RAS amplicons. Band Intensity: The intensity of the bands can give you a qualitative idea of the expression levels. Brighter bands indicate higher expression levels of the target genes.

3. Compare to DNA Markers

• DNA Markers (Last Two Wells): Use these markers to determine the size of the PCR products. Match the bands from the experimental wells to the closest size marker. This helps confirm that you have amplified the correct target sequences.

Interpretation Steps:

Example Interpretation:

• No Bands in Negative Controls (Wells 9 and 18): No contamination. Proceed to analyze the experimental wells.
• Bands of Expected Size in Experimental Wells: Successful amplification of p53 and K-RAS. Note the sizes and intensities.
• Bands in Negative Controls: Contamination present. Need to troubleshoot (e.g., use fresh reagents, clean work area, use new pipette tips).

Actions Based on Results:

• If Contamination is Present:Re-extract RNA, ensure all reagents are contamination-free, and set up a clean PCR environment.
• If Bands are of Correct Size and Intensity:Proceed with quantitative analysis, considering the expression levels of p53 and K-RAS.

Based on the provided gel electrophoresis image and your description, here’s an interpretation of the results:

Observations:

Interpretation:

Recommendations:

By addressing the contamination issue and verifying the results, you can ensure the accuracy and reliability of your RNA expression measurements for p53 and K-RAS biomarkers in colon cancer samples.

thank you all for your detailed answers. We will consider all the comments.

You have contamination. That means you cannot trust any of these data. Throw away ALL (and I do mean ALL) of you reagents. That means the water, buffer, primers, enzyme mix, dNTPS, etc.

Get a new box of tips, clean your bench, get all new plasticware & make fresh solutions of any and all buffer (e.g. TE).

Also, get a positive control sample!

Test out just the positive control and negative controls for each pair of primers until those are working correctly. Then, you can analyze your cDNA samples.

Good luck!

in addition to @Katie A S Burnette answer's : use a new different clean labcoat (not the one you wear when loading PCR products) extract your RNA in a room and with pipettes and material that had NEVER been in the PCR/electrophoresis room. aliquot you primers in a PCR hood with pipettes tube labcoat that are exclusively used for that, use filter tips ... for your PCR use a mix with dUTP than you can get rid of the PCR product treating with dung....

then once you got a negative negative control you can start to interprete the results.