Hello, recently i am an issue swith cloning where I am using the subclone method to insert my gene length is 1.5kb and my vector(pMAL-c6T) is 5kb I got the colonies which i screened through the insert primers for amplifying the gene in the colony PCR i got confirmation of colony where I have the insert amplified. i did plasmid isolation and for another confirmation i did restriction digestion of my insert hasn't appear on the gel only i can vector band and i have repeated the restrction digestion where i got unspecific band at 3kb and vector at 6kb. my insert should be at 1.5kb i did see any band over there
Sometimes you get false positives when doing colony PCR due to the input DNA getting spread on the plate. You might regrow a few dozen colonies after streaking and see if any are positive then screen those.
It is also possible that your 3kb band is a dimer of your insert, given it is 2x the predicted size. Just a thought to investigate.
You can confirm the amplicon size again by doing PCR using the plasmid. You can use different combinations of primers for confirmation. For example, you can use vector-specific universal primers in addition to gene-specific primers, as well as a combination of gene and vector-specific primer pairs, such as one primer that is gene-specific and the other that is vector-specific.
Moreover, check for the presence of restriction enzyme sites in the recombinant plasmid because a 1.5 Kb fall-off will be very clear and sharp on an agarose gel. Check the activity of the restriction digestion enzymes used. You can increase the duration of digestion. If the duration is 1 hour, you can increase it to 2 hours or even overnight. This can help if the enzyme activity has been reduced, which can happen due to several reasons, such as frequent freezing and thawing, improper handling during use, or improper storage conditions.
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