Title: Polymerase Chain Reaction Simulation Propose:The propose of this lab was to understand how by running a gel electrophoresis on a batch of DNA we are able to see how many approximately cycles it has gone through.Methods: Casting the Agarose Gel In this experiment .8% solution was used. By using a 250ml flask the buffer solution was prepared. Using the equation to make enough solution for the entire lab class the equation had to be multiplied by four. The contents of this equation were added to the 250ml flask and swirled to evenly distribute it contents.
Then a mark was placed on the outside of the flask to indicate the level of the solution before heating. The flask opening had perafilm placed over it so that there was little to no evaporation. The solution was then placed in the microwave and heated. The solution was then heated for one min and swirled for evenly dissolved Agarose. The Agarose was then cooled, so that it was not to hot and the plate would crack. Some water was added to the solution because of there was some evaporation during heating. Once the gel had cooled, it was poured into the plate between the rubber dams.
The plate was filled about half way up the comb arms. These dams are placed in the plate to prevent leaking. Then the gel was added and allowed to completely soiditify, which takes around 20mins. Preparing the Gel for Electrophoresis once the rubber dams have been removed (carefully), the comb was then removed. Then the buffer was made.
The buffer was made by using the equation, but also multiplying it by four, for the three lab groups. Then the chambers around the gel plate is filled with the buffer, just enough buffer to cover the gel plate in a very small amount. Then the dyes were loaded to there correct wells. Once the gels were added (carefully) the lid was placed on the plate and system was turned on. The system ran for about 10mins.
(Hint the system is running when there are bubbles occurring in the buffer solution.Once the gel had been run the exactly gel had been removed from the buffer, placed on tin foil and moisten with a small amount of buffer solution. Then the gel had a DNA Instastain sheet placed on top of it.
The sheet was placed on the gel firmly and a beackr and gel casting tray were placed on top of the gel. The stain was allowed to set for two minuets. Then the gel was placed under ultra violet light and the pathways that the different wells made were seen very clearly. This was how the results were accomplished. Results: Due to problems with the loading of the gel, the results of the gel were very inconclusive. The results that should have been observed by the group, are very small line at A (the standard DNA Fragments), At B (Control Sample after 0 cycles) there should have been an extremly small line.
The wells for C (sample after 10 Cycles), D (sample after 30 cycles) and E (sample after 50 cycles) should progressavly get large from one to another. F should be the longest and most easily seen line, because this process was run overnight, which allowed more time for more DNA to be prepared. Questions: 1) Upon reducing with B-mercaptoethanol, total amino analysis of E.coli DNA polymerase 1 indicates the presences of three cysteine residues. How do you rationalize this data based on the information provided?Due to the fact that cysteine is hydrophilic, this leads the results to be that B-mercaptoethanol is also classified as hydrophilic.
2) Why is proofreading by the 3 to 5 exonulcease activity of DNA polymerase 1during DNA synthesis very important?Proofreading is important because it helps find distortion that is caused by unmatched base pairs in the 3 end (which occur between the template and growth chain).3) How does one achieve limited protease digestion?Such proteases like trypin that produces two polypeptide fragments. 4) How are two different primers required for the PCR reaction?One primer is used at each end of the of the DNA strand to produce amplification. 5) Which of the three describe procedures PCR, lCR and CPR is not useful for cloning experiments even though it is useful diagnostic method?LCR