Random Case

Cloning and sequencing chloroplast DNA requires working with sterile objects and attempting to minimize any cross contamination of samples to prevent foreign DNA from being inserted into the experiment. Working in a lab that is exposed to various cells where the ability to be completely sterile is not possible, could potentially contaminate samples and creates an opening for erred results that vary from what the expected should be. Also, without being able to absolutely verify that M. albus chloroplast DNA has been isolated prior to proceeding with polymerase chain reactions (PCR) it is impossible to determine with certainty if M. albus chloroplast DNA was successfully amplified during PCR versus foreign DNA being amplified. With the understanding that the chloroplast genome of a flowering plant is approximately 150,000 to 500,000 base pairs (bp) in length, and knowing the restriction enzyme being used in the experiment is EcoRI (a six base pair "cutter"), it is logical to theorize that the results on the gel electrophoresis photograph, in the row containing successfully inserted M. albus chloroplast DNA into vector pUC18 after PCR with M13 primers, there should be a band present at approximately 4217 bp (Harrison 2012). This would indicate that the M13 section of the pUC18 vector, which is approximately 121 bp in length, includes at least one section of inserted M. albus chloroplast DNA. The length of the M13 including M. albus DNA could vary because it is not guaranteed that the six base pair sequence unique to EcoRI occurs once every 4096 bp. It is possible there are additional EcoRI sites on the M. albus chloroplast genome which would result in varying fragment lengths from the expected and different frequencies of the fragments.