I perform long range-PCR of a 9kb fragment and purify the 9kb band from a gel. However after doing multiple BP reactions, i keep getting a smaller 3kb fragment that is in my plasmid, and no 9kb fragment. Can anyone help me why this is happening and what I can do to prevent this?
Both the ligation and cloning efficiency are reduced as the DNA that you are inserting increases, however 9 kb is not excessive. If you are using a polymerase blend to do your PCR, you can do TA cloning, however if you are using a proofreading polymerase, you will either have to do blunt end cloning (which will decrease your efficiency further), or add an A after cleanup. Before doing the ligation, I would clean up the insert and vector (phenol/chloroform, 2x chloroform and ethanol precipitate) and determine the DNA concentrations, the more accurate the concentrations, the more likely the ligation will work. As you will have low efficiency I would then recommend that you use electrocompetent cells and electroporation as you will get higher transformation frequencies with large plasmids.
Have you checked where your Primers anneal to the vector? It sounds to me that they bind to other sequences which are on your plasmid.
If that's the case I would suggest:
1. Double-check your annealing temperature. Your anneling might be improved if you change your temperature.
2. Check if there are other primers you could use. The possibility of your primers binding to an unspecific sequence can be decreased.
Recombination events with larger inserts is not uncommon.....is the 3 kb sequence always the same? This would suggest a sequence specific artefact that is driving the recombination event and if so it's no simple task (without changing the method you are using) to optimise the reaction to reduce recombination. If this is the case ensure you transform into a recA- host.
You could try cutting your vector between the attB sites then perform a PCR using your insert to prime from. This of course would only work if the sequence on the insert outside the attB sites (both 5'' and 3') was complimentary to the vector and not just random filler.
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