I am trying to clone ~3.8kb insert into a ~4.7kb plasmid vector. Things are fine upto transformation. However, during screening when I am isolating the plasmid, I am getting only one band(sometimes 2). Also this single band is larger than the clone is supposed to be. Further, when I am doing diagnostic digestion with the plasmid, I am getting a single band after double digestion. Can anyone help me with the troubleshoot please?
Hmm, could you post a map and/or general cloning strategy? That way we can help troubleshoot your diagnostic digests. What happens when you use each enzyme individually?
When single digestion is done I am getting single band, although the size is way larger than it is supposed to be.. Sometimes I am even getting ladder-like multiple bands as well. Amy Klocko
The number of bands you obtain after molecular cloning can vary depending on several factors, including the cloning method, the number of restriction enzyme sites in the vector and insert, the efficiency of the cloning reaction, and the number of copies of the plasmid in the bacterial cells.
In an ideal scenario, after a successful molecular cloning, you would typically expect to see two bands on an agarose gel: one representing the original, uncut plasmid (supercoiled form) and another representing the plasmid with the insert (linear or open circular form). This assumes that the restriction enzyme used for cloning has a unique recognition site in both the vector and the insert.
However, there can be variations. For example, if the vector has multiple restriction sites and can undergo partial digestion, you may see additional bands representing different forms of the plasmid. Incomplete digestion or contamination can also result in unexpected band patterns.
It's important to analyze your results carefully and consider factors such as the expected size of the fragments based on your cloning strategy. If you observe unexpected bands, troubleshooting may be necessary to identify and address any issues in the cloning process.
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