I want to clone a gene fragment of around 480 bp in the p-RSET-A vector. To do this, I designed two cloning primers that contain the Bamh I and Hind III restriction sites. The primers worked perfectly in the PCR reaction. Subsequently, I performed a double digestion with the enzymes Bamh I and Hind III and carried out the extraction of the band from a low melting point agarose gel. The vector received the same treatment. I performed the ligation reaction at a 1:3 molar ratio and still got the same number of colonies on the V+ control plate as on the V+insert plate. When I performed the restriction and PCR analysis on the colonies on the v+insert plate, none of them were positive. I clarify that in the design of the cloning primers the addition of 6 bp was taken into account to weaken the restriction sites. Can someone help me?
Can you provide more information on how much vector and insert DNA you added for the ligation reaction? Often, the issue is with this molar ratio only. I use the NEBioCalculator to figure out how much insert and vector DNA to use, based on the sizes of both. Read a bit more about that, and you'll understand. If you're using NEB T4 DNA ligase, there are 2 protocols: one is 1hr at room temperature, and the other, which I prefer, is at 16°C overnight.
Also, the purity of your insert and vector DNA matters. Can you share which kit or method you used for the cleanup of digested PCR products or vectors? Are you sure the restriction enzymes worked perfectly? Did you have a positive control to ensure both enzymes are working?
We can find or make errors at any step of cloning process. If you are new to this, take help from someone from your lab. But still post as many questions you want here. I will help you. The below link might also help you.
Ligation optimization (bitesizebio.com)
Usually 2-3bp after the restriction site is enough. Have you checked the age of your restriction enzymes?
Hind III is not the greatest enzyme to use when cleaving close to the end of a DNA fragment (your PCR product) and it requires a long digest time (~O/N) to even be somewhat efficient. So if you only did a short digest with Bam HI and Hind III, it is likely that most of your insert only has a Bam HI end and is the reason you are only seeing colonies containing empty vector.
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