ABI377 Sequencing of PCR products - (Jun/07/2002 )
I have great success manually sequencing PCR products with the USB Terminator Cycle Sequencing kit. I would like to switch over in some cases to using the ABI377 automated sequencer at the center on campus for some of these products, but I have had very poor results with it. The signal strengths are weak and the calls are difficult to make with high background. Does anyone sequence PCR products on a regular basis that can give me some suggestions for improved results? I normally sequence my products with the same primers used for amplification - I have not tried nesting sequencing primers. I have also used the TOPO TA kit and have had mixed results with sequencing those products as well. All the products are cleaned up with either Qiagen or Bio-Rad kits, so I don't think contamination is a problem.
I must be the only one who reads this board who sequences PCR products. Thank you for all your help everyone!