No or very weak band using 16s primers - (Jun/23/2012 )
Hallo everyone, i an aiming in sequencing bacterial DNA, so i fid my extraction and subjected to 16s PCR , but in many cases i don't get any band and in few cases i got very faint band, am loading only 5 microliters of the pcr product on the gel, do u think i should load more? Or its better to increase the amount of template from the bigining?? Am thinking of doing nesting pcr, but i don't know how to do and how much to take of my previous pcr product?? I realy need the procedure pf nesting pcr as well ad touch down pcr, am new in this field and i need detailed information for beginers from zero please. By the way i got also some non spedific bands and diamers too
A likely problem is TOO MUCH template, not too little. Try 10x and 100x dilutions of your template. You should not need to do nested PCR for this.