I have problems double digest With my newly arrived primers, I amplified the gene I transferred into pGEM-T-Easy vector by PCR. At the same time, I amplified the corresponding gene from wheat DNA. After electrophoresis, I cut the gene region of interest and purified from agarose gel (figure 1). I cut these products with PstI and EcoRI restriction enzymes . my product is 823 bp but I got both 823 bp and 1200 bp after cutting. There is a problem but I don't know. What are the bands I marked in figure 2?
I see a product that same size in your "not cut PCR product from pGEM-T (PstI and EcoRI)" lane. It looks like it's more concentrated in your digested samples.
Do you need to determine the identity of the "unknown band" or can you just cut out the bands you want and do a gel extraction?
The pGEMT easy vector with your cloned gene and digested was used as a template and PCR amplified with gene specific primers. The product loaded on gel shows 2 bands and as your PCR product still has template in lower amounts, which is linearised vector and your 823 bp gene. The other band is the linearised band of your vector and non amplified, while your gene which is amplified is a bright band. This is not an issue.
Typically, off-target DNA bands are caused by either partial digestion or Star Activity. You need to compare your digestion to the expected DNA banding pattern. If the bands in both lanes are similar to the expected pattern and the additional bands are limited to spaces within the upper and lower bands of the expected pattern, the digestion is incomplete (partial). In this case, you may need to purify the DNA to remove any contaminants, use more enzymes, and/or increase the incubation time to ensure complete digestion.
https://international.neb.com/faqs/2016/04/07/why-do-i-see-additional-dna-bands-on-my-gel-after-a-restriction-digest
Also, regarding the probable DNA contamination, check the enzyme, reaction buffers, and gel loading buffer for DNA contamination.
Please refer to the link below:
https://worldwide.promega.com/resources/guides/nucleic-acid-analysis/restriction-enzyme-resource/
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