Paraphrasing of PCR methodology Essay Example | Topics and Well Written Essays - 1000 words

Paraphrasing of PCR methodology - Essay Example This was followed by further addition of one volume of phenol into the tube before centrifuging at 5000 rpm at room temperature. The phenol was obtained by saturating it with NTE of pH 8.5. The next step involved removal of the upper phase and putting into a separate microcentrifuge tube and adding one volume of chloroform:isoamyl alcohol mixed at a ratio of 24:1. The mixture was then centrifuged for 10 minutes at 5000rpm. The last three procedures were conducted (X4) making certain that no material remained at the interphase. The preparation was then removed and 2 volumes of 100% ice cold ethanol added following a previous addition of 1/20 volume of 3M sodium acetate. This was then centrifuged again at 3000 rpm for 5 minutes to obtain DNA pellets that were left to dry. Later, they were suspended in a 50Â µl TE buffer such as 10mM EDTA. This allowed for quantification and assessment of the purity of the DNA using a nanodrop spectrophometer 2000C series. The first step involved calibration of the Eppendorf and Sartorius Biohit pipettes. This was done to ensure that they provide accurate readings. The actual preparation of the mixture entailed adding 2.5ul of 10X ionic buffer, 0.5ul dNTPs, 0.75ul MgCl2, 0.5ul GAPDH forward primer, 0.5ul GAPDH reverse primer, 0.1 Taq, 18.15ul molecular water and 2ul of DNA Template designated THP1. Each 0.2ml PCR tube was filled with 25ul of the supermix. The supermix, DNA template and the primer values of the consecutive experiments 2, 3,4,5,6 and 7 will be changed to obtain the optimal condition of the PCR detection of the PTEN gene. Similarly, in the other experiments, PTEN and GAPDH will be the primers, which will use different cell lines such as HACAT, MM6, Hela, Caski and THP1. The PTEN and GAPDH primers are product of the SIGMA-ALDRRICH company, whereas the cell lines were obtained from the American type culture