Hello. I am working with C. elegans, and I need to design primers that have sticky ends. I already design my primers, but can't figure out how to make the sticky part. I want to insert a DNA region in a plasmid. I've used BamHI, but my PI told me that it resulted in blunt end. How can I make it sticky?
I think you should design primers to amplify your gene of interest. These primers must contain the restriction enzyme sequence which will be restricted by enzymes that will give you sticky ends. After this, perform the restriction reaction and you will have sticky ends
Firstly you design your primers with a complementary base pair sequence to your gene of interest. This sequence should be at the 3' end of your primers. Next to this place your restriction site so that the restriction sites flank your gene. Following this place a short extender sequence.
What restriction site you choose is dependent on what vector you are trying to clone your gene into. BamHI should produce a 4 base pair overhang i.e sticky end.
I hope that helps.
Design the primers by adding restriction sites to 3' end of the primer. this will add additional 6(about) base pairs to your primer. Be careful about primer dimerization and off target binding of the primer after adding restriction sites. BamHI can produce a sticky end after restriction digestion.
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