Cloning Process Buccal Swabs Or Whole Blood Samples (...

Cloning Process Buccal swabs or whole blood samples (2-5ml) will be taken from a patient with Alzheimer’s disease. This will be transported to the lab immediately for RNA extraction. The ChargeSwitch gDNA/RNA technologies Purification Kits protocol will be used for the DNA extraction. A reverse transcription PCR (RT-PCR) procedure will be carried out using the designed primers for cDNA. The cDNA is obtained from the mRNA and not the genomic DNA. Derived cDNA will not be manipulated to obtain sticky ends, rather it will be blunt-ended. The expression vector will be picked and cut, using the restriction enzyme Sma1. The cohesive ends will then be ligated with the blunt-ended cDNA. This process will be efficient with the inclusion of Oligonucleotide and T4 ligase (Croy, 2000) (4). The vector DNA will then be amplified in Difco LB agar. This will involve incubating the sample for 3hours and checking the OD at 600nm after that time. After an OD of 0.4, it will be appropriate for the procedure. At this point we shall be having competent cells. Competent cells will be then thawed, split in samples of 100 ÃŽ ¼l plus 0.1 ÃŽ ¼l of plasmid, kept in ice for 40min and then at 42c for 30sec, by a process of heat shocking; which allows plasmids to transfer into the cell. The sample will be transfered to a 1ml LB media, put in a shaker at 37c and 300rpm for 20min The bacteria will then be distributed into plates with LB plus Ampicillin antibiotic and incubate at 37c for approx. 12hrs. Colonies