High MW PCR band seen...help needed - (Feb/04/2010 )

I need to amplify out a 1.9kb target for gateway cloning. This sequence has about 1/2 of the gateway sequence on the 5' and 3' ends and normally we use a universal primer that adds on the rest of the gateway sequence and PCR's out the insert. However, the forward universal primer has a 58% homology site in the insert and it binds there and amplifies. I designed a longer "universal" primer for both the forward and reverse directions that adds on the rest of the gateway sequence and has a Tm of 60 (only the part that overlaps the template).

Using this 2nd set of primers at 60 and 62 degree annealing temperatures, I get a product >8kp! It is the only band. When I lower the Tm I get a smear from 8kb all the way down to 0.5kb. I have tried increasing the Mg2Cl from 1.5 mM to 2 and 3 mM with little to no success. The GC content is about 52% on the target..

I cannot make shorter primers because the 3' end of the target has a 7bp hairpin in it (can't do anything about this) and needs a Tm >59 to get amplification (as seen from colony screening).

Any advice?

I increased the MgCl concentration 0.5 mM to 2.0 mM total and now I'm getting a "ladder". It looks like running a ligation on a gel and does not have any discrete bands.

You could shorten your extension time to eliminate the 8 Kb band amplification. This might allow a shorter fragment to amplify, though usually a shorter sequence will be preferred over a longer one, so I'd be concerned that there is something else wrong.

I lowered the time to 45 seconds and I still got the 8 kb piece. All my control lanes are empty and my positive control (another template and primer pair) works perfectly indicating that it isn't a contamination problem. One of my primers introduces a point mutation in the last position but in the past that has never affected my PCR reactions.

I don't know of any polymerase that can amplify an 8Kb fragment in 45 seconds. I think you must either be misjudging the length, or have a very large amount of 8Kb template in your reaction to start with. Try running a gel with a sample of your PCR reaction before you cycle it.

I ran a negative control (everything but the template) loading 10 uL on to a gel and no bands were seen. The 8kb band is something that is occurring during the PCR. I agree that this polymerase should not have be able to make an 8kb band in the time allotted. I am thinking about ordering even longer primers with a Tm of 68ish so I can do a 2 step procedure hoping that this will work.

As phage434 said, the 8kb band could be in your template- so a no template control PCR reaction won't rule this out, and doesn't prove that the 8kb is something occuring during the PCR.
Could you do as phage says and run a PCR reaction before cycling it, or just running an aliquot of your template.
How big is your template? Is it plasmid DNA?

I ran the template and is much smaller than 8kb. It is 4.5kb and even supercoiled it does not run like the 8kb band.