Restriction Map Analysis Lab

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Lab Report #1 – Restriction Map of the pTP1 Plasmid Introduction: The purpose of the lab is to apply methods for isolating and analyzing DNA with the use of restriction enzymes in preparation for analysis of the pTP1 plasmid by agarose Gel Electrophoresis. Restriction endonucleases are enzymes that cleave the sugar phosphate backbone of DNA at specific nucleotide sequences. They are crucial tools required for modern recombinant DNA technology. The way these enzymes work is by “scanning” a DNA molecule (usually 4-6 nucleotides) for a specific recognition sequence and then stops to cut the strands at the specific sequence. The restriction enzyme will continue cutting the DNA strand which is broken into fragments that are measured in base pairs (1000 pairs). The restriction enzymes used to analyze DNA in this lab are xHol and Hind III isolated from Xanthomonas holcicola and Haemophilus Influenzae respectively. Plasmids on the other hand used in the lab can be depicted as part of the genetic structure in a cell in which replicate independently on chromosomes, particularly in bacteria consisting of small circular DNA strands. We will construct plasmid maps based on the fragment lengths determined from our results from gel electrophoresis. To prepare our digestion we will combine restriction enzymes along with our plasmid or lambda DNA into 8 microtubes consisting of loading buffer of various proportions measured in microliters. After incubation overnight, the samples are ready for gel analysis. After digestion, microtubes containing DNA, enzyme buffer and restriction enzyme which are frozen and later loaded on agarose gel to be analyzed by electrophoresis. Through electrophoresis, the separation of the DNA can be carried out by their molecular weight running to the positive end since DNA contains a negative charge. Agarose can be used for analysis with

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