Ligation of two PCR products - (Mar/16/2015 )
Following a mistake, I'm facing a molecular cloning problem: how to ligate 2 PCR products alone, without any vector ?
Is that possible ? If so, which amount/ratio of DNA should I use ? Can I use a regular T4 DNA ligase ?
For more details: I have one PCR product of 152bp and another of 664bp, they are both amplified by the Kapa HF polymerase. I purified each product, and then I digested them with the BseYI restriction enzyme, and I want to ligate them...
If anyone as any advice, they are welcome !
It is certainly possible, it was common practice before PCR to ligate short adapters into vectors to get a desired restriction site.
Assuming that you have isolated the fragments you want (i.e. without the bits you don't want):
I would test a range of different molar ratios of the two, and try to amplify across the two pieces using one primer from each fragment (the ones that will give you the biggest product if the reaction works, i.e. the ones on each end of the combined sequence). This gives you some surety that your ligation has worked, as the PCR shouldn't work unless the products are ligated. In addition, it will (should) give you more of the desired sequence to play with.