I currently receive my sequencing result but i found some problems with it!
get dna sample -> amplified desired sequence by our primers -> run gel to confirm whether the product is correct or not -> if correct, go ahead with Restriction enzyme digestion (plasmid and pcr product) -> run gel -> if correct, go ahead with ligation -> transformtion -> pick colonies -> purify -> RE again (confirm whether the plasmid take up the DNA) -> sequencing
My target sequence should be around 500b.p. However, after ligation, I do the transformation and restriction enzyme digestion and then finally agarose gel electrophoresis again, the bands shown were not at 500b.p. Bands at different position (low M.W. and High M.w.) were shown.
Is there something contaminate the sample?
On the other hand, I would like to ask what is the ideal concentration and OD ratio for plasmid? Thank you!
Edited by helpwithdna, 07 May 2013 - 12:55 PM.