Hi,
I'm cloning a plasmid and want to confirm that the construct is correct. I had 3 options of dual-cutters (one cut inside my insert and one cut outside): ClaI, SpeI and NdeI. ClaI and SpeI didn't result in the correct bands, although the size of the uncut plasmid was correct (around 8kb). I suspect that maybe it has something to do with the fact that ClaI can be inhibited by methylation and the target sites for ClaI and SpeI in my construct are overlapping... With NdeI I got the expected bands for all my colonies, but decided to do another digest to be sure. So, I want to use two single cutters: one that cuts inside the construct (SmaI) and one outside (AscI), but SmaI optimal incubation temp is 25º so I was wondering if I could incubate first just with SmaI, inactivate it and then with AscI. I dont have another option of single cutter inside the insert, so if this is not possible I'll probably have to buy another enzyme.
Yes, you can do that without problem. In fact you don't even need to heat kill if you are just doing diagnostic cuts. However be sure you are using a buffer system where both enzymes work well.
SpeI should not have been affected by methylation, only Cla (sometimes). If you know the sequence at the two ClaI sites you can predict if it is blocked or not.
Have you thought about verifying your clone by PCR?
Firstly, if you have the option, send your construct for sequencing. If not perform a pcr (even a colony pcr will do) to check the presence of your insert DNA. Now if you really want to perform digestion, it wouldn't be a problem if you incubate first just with SmaI, inactivate it and then digest with AscI. As mentioned by Michael J. Benedik, you have to check the buffer compatibility of the two enzymes.
Dear Karina
You can obviously do so as you have asked. Before proceeding with the experiment, check the buffer compatibility of the enzymes. If they are not compatible then after first digestion, purification is recommended. Finally i would suggest that go for PCR check for the presence of insert and then sequencing as digestion and all these will be arduous.
I assume you are using NEB enzymes. you can make a double digestion using cutsmart buffer. use AscI first till 80 percent digestion and then add SmaI and set at 25 degrees and run on gel till you find your desired product. you can still go for sequential digestion as you mentioned. you can design primers for half of the binding seq on insert and other half on vector. its much cheaper than buying a new enzyme. all the best
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