Lab8_Wilkerson - Christopher Wilkerson 1 6/24/08 Ligation...

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Fig. 2 Plate 1, Promoter plasmid only. Fig. 4 25ng Control plasmid. Christopher Wilkerson 1 14:40:36 Ligation and Transformation Objective : The purpose of this lab is to finish the Biobrick cloning experiment by ligating together the two fragments of DNA digested previously, the vector (tetR) and the insert (mCherry). Before ligation can occur the two pieces of DNA must be purified from a gel. Once the two portions of nucleic acid are obtained another gel will be run to verify that the purified samples are of the correct length. T4 DNA ligase will be used to join the vector and insert together. DNA ligase has the ability to use ATP to create phosphodiester bonds between the sugar and phosphate backbone. The base pairing between the sticky S & S ends and the two sticky P ends of the vector and insert bring the two strands close together so that the ligase can effectively join the two DNA strands. Since the vector is significantly larger, the smaller length inserts must be higher in frequency to ensure that the base pairing between the sticky ends will occur. In particular we will need a 3 to 1 ratio of insert to vector. This will have successfully placed our gene of interest, mCherry, downstream of the tetR promoter while conserving all four Biobrick digestion sites. Chemically competent E. coli will then be transformed by this newly formed plasmid. If all goes well the bacteria should be able to produce a protein that will fluoresce red under UV light. Lab Protocol : We first extract the DNA fragments from the gel by using the Sigma GenElute Gel Extraction kit. The two gel samples are massed and an amount in μ L that is equal to 3X the mass in milligrams of the gel is added to the microcentrifuge tubes containing the gel samples. These tubes are then incubated at 55
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Lab8_Wilkerson - Christopher Wilkerson 1 6/24/08 Ligation...

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