Molecular Method and DNA Separation

Plasmid Prep: Lab Write-up
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Plasmid Prep: Lab Write-up
Part one
0475615
Reference gel

• L - ladder
• P - uncut plasmid
• E - EcoRI digestion
• N – NdeI

–     NE double

• D – DraI

–     DE double

• B – BamHI

–     BE double

• H – HindIII

–     HE doubles

• 1+ - GFP1F/R + pGLO
• 1- - GFP1F/R - pGLO
• L - ladder

On the gel provided above, the L – represents the solution of molecules used as a reference in estimating the size of the DNA molecules. Additionally, L in the reference gel estimates the sizes of the DNA molecules that were earlier separated based on the DNAmolecules' mobility in an electrical friend in electrophoresis. Additionally, EcoR1 represents the restriction enzymes employed in the estimation of the band sizes in molecular biology from the reference gel. EcoR1 makes the four nucleotides that ends at 5' and the subsequent overhang of AATT. Additionally, EcoR1 as a restriction enzyme creates a palindrome sequence that means that there is harmony in the representation of the bases when reading the sequences both from back and forward. The EcoR1 restriction enzymes during digestion cuts at G! AATTC in the palindrome sequence (Green et al., 2019). In the gel references, the digestion by EcoR1 restriction was not complete. The incomplete digestion that EcoR1 presented stems from the possible there could have been significant EcoR1 cleavage sites that had not been cleaved. The reason as to why there is a conclusion that the digestion was not complete is because, from the gel reference in E, there are plasmids that are liner while one is coiled. It is important to note that the linear plasmids mean that the digestion was complete but moving along to the positive end, one of the plasmids is coiled, which means digestions were likely to have been incomplete. Additionally, enzyme concentration is the key determinant of indigestion. Additionally, N represents the Nde1 restriction enzymes used in gene cloning to cut open specific target sequences from the reference gel. In the reference gel provided, the digestion was not completed because, first off, the gel visibility is not clear, suggesting that the concentration of the plasmid used was relatively low. For example, when inserting a concentration of 10ng of the DNA. The bands will be faint and not very visible in the gel. Therefore, a major determinant of enzyme restriction digestion is the concentration of the inserts. Consequently, from the reference gel, B represents the BamHI restriction enzyme. The BamHI restriction enzyme binds the at the recognition sequence from 5' 3' direction. BaHI can recognize short sequences that specifically cleaves at a target site (Puria et al., 2019). Additionally, H in the reference gel represents the HindIII restriction enzyme that cleaves in a palindrome sequence as the EcoRI restriction enzyme. In HindIII, the digestion was complete in both the single and double digestion. The bands were also clear in single and double digestion, which means that the enzyme concentration used in the gel electrophoresis was enough to sufficiently complete the digestion. Part Two Question Three   EcoRI:                         1^st insert -between 350bp and 400bp                                     2^nd insert – between 250bp and 300bp BamHI:                       1^st insert – 650bp                 &...