EXPERIMENT 3 (Labs 6, 7, 8, 9, 10, and 11)
Basic DNA cloning
Schedule
Lab 6 -Restriction digestion and gel isolation of DNA fragment -Restriction digestion of vector |
Lab 7 -Set up ligation reactions |
Lab 8 -Transform bacteria with ligation reactions -Run ligation reactions on an agarose gel |
Lab 9 - Pick transformants for DNA isolation |
Lab 10 -Miniprep DNA isolation |
Lab 11 -Restriction enzyme digestion of clones |
Goal: To introduce you to the most basic procedures used to manipulate DNA in Molecular Biology.
Background
First, you will isolate a EcoRI XhoI DNA fragment that carries the Drosophila Sex combs reduced (Scr) cDNA. In addition, you will digest the bacterial cloning vector pUC 19 with EcoRI and SalI. Second, you will ligate the vector with the Scr DNA fragment using T4 DNA ligase. Third you will transform bacteria with the ligated DNA. Fourth, using blue white screening, you will select a putative clone containing the Scr DNA fragment. Fifth, you will determine whether you have indeed identified a clone containing the Scr DNA fragment.
During the experiments you will be monitoring your progress with technical controls, such that if you are unable to identify a clone, you will be able to determine the technical reason for the failure of the procedure. Learning to cope with failure and troubleshoot the problem is an important part of becoming a skilled experimental scientist.
Genotype of DH5a E. coli cells
F- f80lacZDM15 D(lacZYA-argF)U169 recA1 endA1 hsdR17 (rk-, mk+) phoA supE44 thi-1 gyrA96 relA1 l-
Can you tell me what all this means?
Readings
Modern Genetic Analysis 2nd ed. pages 214-225