Dna Restriction Lab Report

DNA Restriction Mapping

Danielle Niemeier [e-mail] BCH 467 Analytical Biochemistry

Lab Section: 16371

Abstract

The purpose of this experiment was to determine whether the vector PRSETB or pQE30 is present and to create a restriction map for the unknown plasmid A. The plasmid A was digested with enzymes BAMH1, PstI, and ScaI and then the resulting fragments were run through an agarose gel via electrophoresis. From the gel electrophoresis and deriving an equation by plotting the log of the size of the DNA size markers and distance migrated, a restriction map was constructed. The restriction map showed that the plasmid has only one ScaI site, which supports that vector PRSETB, is present in the plasmid. From the gel electrophoresis, it

One sample had 250ng of plasmid A as well but with no enzymes added. All the digestions tubes were incubated at 37℃ for 30 minutes. After incubation, 5μL of loading buffer (30% glycerol, 10 mM Tris-HCl, pH 8, 1 mM EDTA, 0.025% bromophenol blue) was added to each sample. 50 ml of molten agarose (1% agarose boiled and cooled to 55℃ with added SYBRsafe) was poured into the casting tray for gel electrophoresis. Once the gel hardened, .5X TBE (44.5 mM Tris base, 44.5 mM boric acid, and 1.0 mM EDTA) was added just until the gel was covered with the TBE buffer. Each sample was loaded into the gel as well as 10 μL of DNA size markers (1kb ladder, New England Biolabs) into a separate lane. The gel was allowed to harden at room temperature and then electrophoresed at 100 volts for 75 minutes. Using a UV imager, a photo was taken of the resulting traveled DNA fragments in the gel.

Results

Table 1. Starting Materials For Single and Double Digestions of Plasmid A BamHI

(μL) PstI (μL) ScaI (μL) BamHI and PstI (μL) BamHI and ScaI (μL) PstI and ScaI (μL) Uncut plasmid (μL)

Digestion Buffer 10 10 10 5 5 5 15

Plasmid 50ng/μL 5 5 5 5 5 5 5

BamHI 2U/μL 5 - - 5 5 - -

PstI 2U/μL - 5 - 5 - 5 -

ScaI 2U/μL - - 5 - 5 5

Each band under the single digestions in Fig 2. indicates the number of restriction sites each enzyme has in the plasmid. In Table 2. BamHI was shown to have cut the plasmid twice at 4.0 kb and .7 kb, PstI cleaved the plasmid once at 4.5 kb, and ScaI cleaved the plasmid once at 4.9 kb. Taking the sum for each band, the plasmid’s size after being cleaved was determined to be 4.7 kb. For the double digestion with BamHI and PstI, there should have been three bands but Fig 2. only shows two- one at 4.0 kb and one at .7 kb (calculated in Table 2). This could have been due to accidently only digesting the plasmid with BamHI rather than a combination of BamHI and PstI. It was also possible that there weren’t enough PstI enzymes to digest the plasmid. To correct this, more amount of enzymes could be added and further care to avoid cross contamination should be used. The double digestion for BamHI and ScaI resulted in 3 bands that were 2.3 kb, 1.3 kb, and .7 kb in size. From this information, it could be discerned that ScaI and BamHI both cut the plasmid that resulted in a fragment that was .7kb in size. The 2.3 kb and 1.3 kb fragments that were created after the double digestion with BamHI and ScaI indicates that the ScaI site was 2.3 kb and 1.3 kb away from the BamHI sites as can be seen in Fig 4. The double digestion for PstI and ScaI resulted in two bands