Restriction fragment length polymorphism - (Apr/01/2007 )
Can any one please guide me on how to calculate how much restriction buffer to use, when a smaller unit of enzyme is required.
For e.g. from a 10u/ul Hinf1 enzyme, the product handbook recommends 2 ul of the enzyme and 2 ul of the buffer. However this will result in 20 unit of the enzyme being used, when many MTHFR protocols only require 8U. how then do i know how much to drop my buffer down by? and should i include water into my restiction protocol, cos as it stands im adding 10ul of the pcr product+ 16ul of water, 2ul of Hinf1 enzyme and 2ul of Buffer.
Can any one assist
Most (all?) RE buffers come in a 10X concentration. That means you have to dilute the stock buffer 10X in your reactions to end up with a 1X concentration. So if your reaction volume is 20 ul, you need to add 2 ul of 10X buffer. If your reaction volume is 68.4 beer mugs, then you need to add 6.84 beer mugs of 10X buffer. Adjust the amount of water in the reactions to bring the total volume to whatever you want. In the example you've given, your total volume is 30 ul, but you are only adding 2 ul of buffer. This is not enough. Add 3 ul of buffer and reduce the water to 15 ul. Also, remember that your PCR reaction contains salts that affect your reactions. You may need to experiment to find what works best. Cheers.