questions about the agarose bead protocol - (Jul/09/2006 )

Hi,all

In Labtechie's post ,he suggesed one bead per PCR reaction.I wonder what kind of PCR volume is appropriate,20ul,25ul or 50ul? And how many Mg2+,primers and dNTP should be used?

Another question is that there is always a little water adere to the agarose bead when I pick it out of EP tube.It's difficult to remove.Will the water affect the PCR result ?Or I can just overlook it?

Wish people who success with agarose bead protocol could give me advice.Thanks in advance!



rosylight

Hi Rosylight,

I've had luck with the agarose bead PCR method.

In the first round of PCR where the bead is your template, I used a 100ul PCR reaction. I think it would be the same as a 25ul reaction, only upscaled (apart from the volume taq maybe). The main reason for this was that the bead made the reaction 'thick' and after a 100ul reaction, the viscosity of the reaction is less. As to the actual volumes of Mg and primers, I'd guess that would depend on the reaction, but I have listed below one which I have used. No doubt you will need to tweak this for your particular reaction.

water 63ul
10x buffer 10ul
dNTPs (2.5mM) 8ul
MgCl2 3ul
Forward (20uM) 4ul
Reverse(20uM) 4ul
Taq 0.8ul

I'm sure I got this from a reference somewhere, check the other posts regarding the bead protocol where someone mentions it.

Best of luck,
Dave