Locating The Xhoi Recognition Site On Lambda Dna Using
1704 WordsFeb 15, 20177 Pages
Locating the XhoI Recognition Site on Lambda DNA Using a HindIII and XhoI double digest
Restriction enzymes cut DNA at certain sites to create multiple DNA fragments. Restriction enzyme HindIII has known DNA fragment lengths and recognition sites when digesting lambda DNA, while the lambda DNA recognition site for restriction enzyme XhoI is unknown. The goal of this study is to determine the lambda recognition site of XhoI by comparing a HindIII digest and a HindIII and XhoI double digest on an electrophoresis gel. The HindIII digest had a band at 9.4 kb, but this band was not visible in the double digest, therefore we concluded the recognition site for XhoI was around 9.4kb. There were also two additional DNA…show more content…
XhoI is a type I restriction enzyme, so the location of the recognition site on lambda DNA is unknown but it is known that it cleaves at CTCGAG sites (XhoI (10u/ul)). While the DNA fragment lengths and cleavage sites are known for HindIII lambda DNA digest, they are not known for a XhoI lambda DNA digest. The goal of this study is to determine the recognition site of XhoI on lambda DNA by comparing the DNA fragments from a HindIII digest to the DNA fragments of a HindIII and XhoI digest. We will do this by creating a mixture of solutions containing only DNA, DNA and HindIII, DNA and XhoI, and DNA, HindIII and XhoI. We will then run a gel electrophoresis, which will separate the DNA, fragments by size and we will compare the DNA fragments from each solution. Through comparing the DNA fragments from the HindIII digest and the HindIII and XhoI double digest, we will be able to determine the XhoI recognition site on the lambda DNA.
Preparation of restriction digest solutions:
To begin the process to determine the XhoI recognition site in the lamda DNA fragment we first prepared 4 tubes of solutions containing 10X Optizyme reaction buffer, sterile water, lambda DNA (0.3 ug/ul), XhoI (10u/ul, 3000u), and HindIII (10u/ul, 7500ul). Tube 1 contained 2ul 10X Optizyme, 16ul sterile water, and 2ul lambda DNA. Tube 2 contained 2ul 10X Optizyme, 14ul sterile water, 2ul lambda DNA, and 2ul XhoI. Tube 3 contained 2ul 10X Optizyme, 14ul sterile water, 2ul