Gene Therapy: Lab Report

The LIEU component Is a linear disperse that does not notation an Autonomous Replication Sequence, so it could not reprize on its own and needed to be integrated by homologous recombination. The TRIP gene was a circular plasmid that contained an EARS, which allowed for it to act as an extra chromosome In the gene. The objective was to insert a wild gene and replace the defective genes and then grow them on a sensitive that does not contain TRIP or LIEU to prove that the genes had been cured.To tending determine if recombination took place in the LIEU gene, and to compliment negative entropy from the 431 LIEU drop out medium, the cured LIEU gene was compared to the unhealthy ELISE gene. The expectation was that the cured LIEU gene would be a dissimilar size from that of the diseased, which would be proven through a PC thrash of the two DNA strands after they were replicated under the same in vitro conditions. The adjudicate of the PC was to show what kind of mutation occurre d in the mutant to actor It to lose Its LIEU function.Methods Yeast Transformation Procedure Both work force and bench tops were sterilized by 10% ethyl alcoholic beverage and were continually wiped down at various times throughout the lab. Gloves were also worn for the duration of the lab to help prevent contamination. The first amount was to obtain doth strains of yeast, AY 235 and AY 431, with the fat end of a sterile tooth pick from an call plate and place them into two separate Offender tubes.The Offender tubes were alter with poll of solution 1 (50 ml sterile water) before the yeast was added to them. The tubes were then spun in a extractor for four seconds to separate the excessiveness water from the pellet that formed from the yeast. The supernatant were discarded and the pellets were suspended in poll of solution 2 (0. MM Lilac 0. 01 M Tries, 8. 0 0. MOM DEED). The solution was once again spun for four seconds in the centrifuge and the supernatant were discarded. Th e pellets were re- suspended in 1 Pool of solution 2.