I am a PhD student and stuck with the no band in the gel results.
I am using the following
- 1 microliter Primer R (20mM)
- 1 microliter Primer f (20mM)
- 1 microliter dNTP (10mM)
- 1 microliter DNA Template ( 448 ng)
- 1 microliter TAQ polymerase
- 5 microliter Buffer (10x) added MgCl2 by 1:1
- 30 microliter PCR grade water
- 95 for 4 minute
- 95 for 45 sec
- 55-65 for 1.20 minute
- 72 for 1 minute
- Gradient 35 cycle
- 72 for 4 minutes
110 volt, 200 mPA for 30 - 45 minute
I get PCR products as in the images. There are no bands while there are intense bands at the bottom and some of the wells.
My question is, is that at the bottom products?? what can be done to make them real? If not then why there are differences between the two lane of wells. Each 12 well are representing a one type of DNA template (I used three different organisms DNA).