purified dna using dh5alpha and qiaprep....plasmid not in genome - (Apr/16/2010 )
So I'm a chemist and new to the bio world. I just started using biotechniques to try to make some mutant proteins. I was given some transformed DH5alpha with ampicillin resistant plasmids and purified using qiaprep spin miniprep kit. I submitted the dna to the genome center and none of the samples contained any of the desired genes. However when i digested them and ran them through aragose gel comparing them to the plasmid that was used, the single and double digested strands matched up while the undigested strands did not. Anyone know what's going on? I am clueless
Undigested plasmid is circular, and can often be supercoiled (wound like a badly managed phone cord). These forms run peculiarly on a gel, often faster or slower than the corresponding linear fragment of the same size. Often, multiple forms are present, yielding several distinct bands, even though only one sequence is present.
are you saying also that the plasmid was taken up, even though it is found nowhere in the genome?
Your plasmid has ampicillin resistance gene and DH5alpha doesn't. So the fact that the cells can grow on selective medium containing ampicillin means that the plasmid has been taken up by the cells.
When you search your sequencing result for your target sequence, depending on the sequencing primer's position and direction, you might get reads on only partial sequence or the complementary sequence. Make sure that you don't miss it because of those. Use blast2n tool to compare your sequencing result to your target sequence.