. Each PCR reaction is specific for the target cDNA you wish to amplify. Thus, i
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Question
. Each PCR reaction is specific for the target cDNA you wish to amplify. Thus, it is often necessary to “optimize” a PCR protocol for each target. Review the procedure for today’s exercise and state any points which could be adjusted to improve results.
Procedure:
Week three: From transformation to expression and phenotype
Techniques: Screening of bacterial cultures, RT-PCR and gel electrophoresis
Specially prepared tubes, which contained the buffer, the dNTPs, and then enzymes as a white pellet were used to begin this section of the experiment. It was endured to Be very careful with these tubes, as they were very expensive and replacement tubes were not available. It was absolutely critical that gloves were worn when handling all samples. All reagents and samples were also kept on ice at all times!
Four RT-PCR tubes (0.5 mL micro centrifuge tube with a white pellet in the bottom) were obtained from the TA and labeled as follows:
– Control (No RNA)
+ Control (GFP transcript)
RNA isolated from LB/Amp Colonies
RNA isolated from LB/Amp/Ara Colonies?
17 ?L of room temperature H2O was added to each tube – being sure to NOT touch the pellet with the tip. The lid was then closed and each tube was gently taped on the table so the H2O rolled down to the bottom. The tubes were then placed in the ice bath for 5 minutes.?Using the table below, the following reagents were added to each of the tubes:
The tubes were then taken to front table on ice and each tube was vortexed for 1-3 seconds. Then, the tubes were centrifuged briefly in the micro centrifuge to collect fluid droplets in the bottom of the tube. The tubes were then immediately returned to ice bath, until the instructor was ready to place them in the thermal cycler. The program used for this experiment was 95 oC for 45 s. (denature); 56 oC for 45 s. (anneal); 72 oC for 45 s. (elongation). This was then run for 25 cycles.
Near the end of the thermal cycler run, the agarose gel was prepared. the gel tray was then set crosswise onto the raised portion of the electrophoresis tank, and gentle pressure was used to fully seat it. The agarose solution (agarose, buffer, ethidium bromide in the already prepared flask) was then heated in the microwave for 30 seconds (until all of the agarose melted). It was ensured to wear gloves for this experiment due to the fact that ethidium bromide is extremely dangerous, and the solution was boiling hot. The solution was then cooled with gentle swirling for 1 minute. The agarose was then poured into the gel tray, and the comb was placed into the appropriate slot in the tray. The gel was then allowed to cool completely, and was then lifted out of the tank, and rotated so that the comb end was towards the black electrode, and set back into the tank. The tank was then filled with running buffer until the gel was just slightly covered. Then, the comb was gently removed, straight up.
After the thermal cycler finished, 5 ?L of loading buffer was added to each tube. Each tube was then briefly vortexed and placed in ice bath.?
6 ?L of DNA size standard was then added into the first well (‘lane 1’) and 20 ?L from each sample tube was added to the next wells in the gel. The samples were then loaded in the following order: Size Standard, Negative Control, Positive Control, LB/Amp colonies, and LB/Amp/Ara colonies.
The gel was then run at 150 Volts for 30 minutes. The gel was then Visualized and photographed using the UV Transilluminator and a camera, making sure to wear safety face shields to ensure personal safety.
Sample Random Primer Primer LB/Amp LB/Amp/Ara Hexamer (Forward) (Reverse) Control Control RNA RNA Control 1 HL 1 uL uL 5 uL +Control 1 uL 1 uL 1 uL 5 uL LB/Amp 1 HL 5 uL 1 uL 1 uL 5 uL AraExplanation / Answer
In the given protocol the following points can be adjusted for improvement of results:
Section 2:
Section 3:
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