PCR Lab- 08/31/16
In this lab, our objective was to mix all of the components to complete PCR. Before beginning, we reviewed how to use a micropipette in order to achieve accurate results.
Observations
The final mixture for our PCR reaction required a final volume of 50 μg, however, some of our reagants were necessary in amounts that were ouside the appropriate intervals for our micropipettes. For example, we needed to mix 0.5 μg of DNA polymerase, but our micropipette was designed in the range of 2-20 μg. Since the four different groups each had the same irregular reagents in common, we decided to create a master mix of the nonspecific components of the reaction to share between the groups.
To do this, we mixed five times the necessary amounts of GC buffer, dNTPs, and DNA polymerase in order to distribute across the groups and have extra in case of human error. When the mix was completed, we noticed that there were bubbles in the solution, so we decided to centrifuge it to remove them. Afterwards, the master mix was distributed between groups who each proceded to add the reagents specific to their genotype.
Method
Our process for making the PCR mixture followed the protocol with a bit of modification (use of the master mix). Additionally, reagants should be kept on ice at all times.
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The necessary volumes of each reagant specific to our group and the TOS3 gene were as follows:
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10 μM Forward Primer (2.5 μl)
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10 μM Reverse Primer (2.5 μl)
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10 mM dNTPs (1 μl)
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5X Phusion HF Buffer (10 μl)
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Phusion DNA Polymerase (0.5 μl)
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Template DNA* (2.0 μl)
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Nuclease Free-Water** (31.5 μl)
*Template DNA volume determined by concentration (~99.9 ng/μl)
**Water volume determined by the amount needed to obtain 50 μl of mixture
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However, a master mix of ingredients common to each group was created separately:
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5 μl dNTPs
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50 μl Buffer
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2.5 μl DNA Polymerase
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IMPORTANT NOTE: When using a micropipette, always discard the tip when moving to a new substance. Use a clean tip for each different substance to avoid contamination.
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The method for mixing the reagants was as follows:
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Mix the master-mix components one at a time using a micropipette into a PCR tube.
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Micropipette the master-mix up and down to thoroughly mix it.
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If excessive bubbling occurs, centrifuge the master mix for 10 seconds to settle it.
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Add group-specific reagents (primers, template DNA, and water) to a separate PCR tube using a micropipette.
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Micropipette 11.5 μl of master mix into the group-specific PCR tube (total PCR mixture volume should be 50 μl).
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Micropipette the mixture up and down to thoroughly mix it.
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Flick or tap the side of the tube if any mixture remains stuck on the sides.
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Place PCR tube with 50 μl of mixture into a programmed thermal cycler and run the reaction.