Suggestions needed for PCR

[Ramon 'Jammin' Cuevas]

Hi. I'm currently a "beginner-student" learning the fundamentals involved
with research in molecular biology. Just recently I learned the basics
involved with the polymerase chain reaction (PCR), and its applications
in amplifying specific DNA fragments. My first few attempts were fairly
successful (running gels confirmed this). But recently, I've been having
problems trying to amplify a much larger-sized insert. My question is this:
can anybody tell me what "variables" can be modified that can possibly
improve amplification (ie. changing the amounts of certain reagents; adding
"catalytic" reagents; changing the number of cycles). In case this information
is needed, the "recipe" I've had much personal success with includes the
following:
		1 (x2) uL of each primer
		0.5 uL of Amplitaq (DNA polymerase) enzyme
		5 uL of PCR buffer
		8 uL of dNTP solution
		1.5 uL of MgCl2
		2 uL of DNA template
		32 uL of water

		94oC for 45 sec
		45oC for 45 sec
		72oC for 2 min

Any help (no matter how trivial) is most greatly appreciated.  :-)


Ramon F. Cuevas
rcuevas at umaxc.weeg.uiowa.edu