Optimizing Condition of PCR Detecting PTEN Gene in DNA - Essay Example

OPTIMIZING CONDITION OF PCR DETECTING PTEN GENE IN DNA Optimisation of PCR condition for detecting PTEN gene obtained from Human cell linked with senescence and ageing Extraction of the DNA The university laboratory personnel assisted in extracting the DNA from the six cell lines. Nearly 10 million cells were washed for five minutes in a two cycle process using a combination of 100g pellets and PBS. This was followed by immersing the cells in a 1ml NTE solution made up of 10mM Tris pH 7.5, 1mM EDTA, and 100 mM NaCl. Subsequently, a proteinase K/NTE preparation obtained from mixing 1mg/ml of proteinase K in NTE solution was warmed at 37oC for 5 minutes before adding 100µl of the preparation to the cell suspension. A final solution of 0.5% w/v concentration containing Sodium dodecyl sulphate (SDS) was incubated overnight in a water bath maintained at 37oC. 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 50µl TE buffer such as 10mM EDTA. This allowed for quantification and assessment of the purity of the DNA using a nanodrop spectrophometer 2000C series. Preparation of the Supermix 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 collection (ATCC) or the UK based European Collection of Animal Cell Culture (ECACC). The primers were obtained through a BLAST search engine http://www.ncbi.nlm.nih.gov/pubmed and acquired from Sigma Aldrich in Dorset UK. On the other hand, the 10mM dNTP mix, 50mM MgCl2, 5 units/ul Biotaq polymerase and the 10XNH4 reaction buffer was a product of Bioline. New England’s Biolab provided the 100 base pair ladder that was used in the experiment. The TBE and loading buffers were produced by Fisher scientific. Amplification of the DNA using the thermal cycler A C1000 thermal cycler produced by Oxford’s, UK-based BioRad was used for DNA fragment amplification. After preparation of the sample slides and placing them in the thermal cycler, step 1 entailed heating the thermal cycler solution to 95oC for approximately 5 minutes. For the second step, the solution was heated at 95oC for 1 minute. Afterwards, heating of the solution was reduced to 30 seconds at 56oC, before increasing the temperature to 72oC for 1 minute in step 4. Steps 2 through to 4 took a minimum of 35 cycles after which the temperature was maintained at 72oC for a total of 7 minutes. This was followed by cooling the solution to 12oC for the remainder of the time. Finally, the samples were allowed to remain in the thermal cycler for 2 hours with the temperature maintained at between 56oC and 66oC before removal. Thereafter, 5ul loading buffer was mixed with the sample using a pipette at a ratio of 5:1 (1ul loading buffer for 5ul of sample). Altered parameters In the optimization of the PCR condition for detecting the PTEN gene involving the HACAT cell line, the supermix recipe for experiments 3, 6 and 7 were altered. However, a similar method was applied in all the experiments. Experiment 2 used the basic recipe for supermix with the HACAT cell lines and PTEN at a temperature range of between 56oC and 66oC to determine whether or not a product is formed. The altered parameter in experiment 3 was the MgCl2 concentration that was changed to obtain the optimum magnesium concentration. MgCl2 concentration was varied as follows; 2.25ul, 2.00ul, 1.75ul, 1.50ul, 1.25ul, 1.00ul, 0.75ul, 0.50ul, 0.25ul. Each of the different concentration was added to one of the nine different sample tubes. Since it was important to have a total of 25ul in each of the sample tubes, after adding the MgCl2, molecular water maintained at 62oC was added to compensate for the remaining amount. The recipe of the supemix and the method remained the same in experiment 4, but the temperature gradient ranged between 56oC and 60oC. The temperature for experiment 5 ranged between 60oC and 70oC; however, new range parameters were introduced such as Taq, dNTP, MgCl2 and ionic buffer. In addition, experiment 5 used a new Hacat template to ensure the new parameters work. Experiment 6 entailed altering the DNA templates from the HACAT to THP1, Hela and MM6. Each DNA template is maintained separately at a temperature gradient between 60oC and 70oC. The Last experiment maintained the same method and supermix recipe using the GAPDH primer ay 59oC. Gel Electrophoresis and Transillumination of UV The gel for running the gel electrophoresis was a combination of 30 ml TBE buffer and 0.6g of 2% agarose that mixed in a flask. To dissolve the particles, the mixture was microwaved at 700W. The solution was then allowed to cool before being poured in the MSMini gel electrophoresis tank. This allowed for making for the wells for the samples using a comb. TBE buffer was used to cover the gel after solidification and loaded into the wells. 10ul of each sample and 6ul of the ladder would be required for insertion if 8 wells are obtained, whereas 8ul of each sample and 6ul of the ladder would be required for insertion in each well after 12 wells are obtained. A 100 bp ladder was used. Running the gel electrophoresis requires plugging the CS-300 Geneflow to an electric source and adjusting the current to 700mA at 100 volts for 60 minutes. At the end of the 60 minutes, Ethidium bromide, obtained from sigma Aldrich, is used to strain the gel for about 15 minutes using a Stuart scientific orbital shaker. Afterwards, water is used to disdain the gel for 5 minutes before photographing under UV trasillumination. Read More