help with pcr cloning

[Sailesh Surapureddi]

Hi
	Though not a wizard, had figured out why PCR doenst work in some 
cases. In your case
1. You are using too much of template, reduce it by ten fold i.e take 
20ngs only.
2. Pls see that there are no cholarmines and DEPC in water, both of 
these things inhibit PCR, even if you are using Mill Q, water, these 
two tend to come along, so  the best way out is to use autoclaved 
water.
3. You can take the PCR mix which you already have in 1:100 dilution 
and use that as template and you get your good fat band of your 
intrest.
4. Last but not the least, if you have repeatedly thawed your samples 
from your freeezer in trying to figure out why your earlier PCR did 
not work, then its better to denature them at 95 C for 10 min and then 
use them, alternatively the dNTPs should be prepared freshly.
Hope these help you out in PCRing.
Ciao
Sailesh.