Title: Amplifying a specific gene through PCR
Course: BTEC-1100-301-302-303-F21 > Section: Module 3 - Polymerase Chain Reaction

Protocol for PCR

Stock Reagents for PCR

- 12.5 μM forward primer (BGLALICFWD)
- 12.5 μM reverse primer (BGLALICREV)
- 200 ng/μl template DNA (B. Halodurans Genomic DNA)
- 10X PCR buffer
- 25 mM MgCl_2
- 2.5 mM dNTPs
- 1 unit/μl of Taq polymerase

Method

With the stock reagent concentrations as above calculate the required volume of each reagent necessary to make a 50 uL of the PCR reaction. The reaction concentrations should be: 0.25 μM forward primer, 0.25 μM reverse primer, 200 ng template DNA, 1X PCR buffer, 1.5 mM MgCl_2, 0.25 mM dNTPs, and 1 unit of Taq polymerase.

The cycling conditions should be:

- 1 cycle at 95 ºC for 5 minutes
- 25 cycles of incubations at:
  - 95 ºC for 30 seconds
  - 50 ºC for 30 seconds
  - 72 ºC for 1 minute
- 1 cycle at 72 ºC for 5 minutes

In the PCR lab you will be using the following sheet:

Reagent Stock concentration | Reagent Final Concentration/Amount in Reaction Mix | Volume (uL) of Stock Solution Required to Make Reaction Mix
12.5 μM forward primer (BGLALICFWD) | 0.25 μM forward primer (BGLALICFWD) |
12.5 μM reverse primer (BGLALICREV) | 0.25 μM reverse primer (BGLALICREV) |
10X PCR buffer | 1X PCR buffer |
25 mM MgCl_2 | 1.5 mM MgCl_2 |
2.5 mM dNTPs | 0.25 mM dNTPs |
200 ng/μl B. halodurans genomic DNA | 200 ng |
1 Unit/μl Taq Polymerase | 1 Unit |
water | — |
Total Volume | — | 50 microliters

A positive and negative control should also be run with your PCR. A positive control is a reaction that test for the quality of our work and reagents. It is a reaction that has been performed with defined results. If the positive control reaction is performed correctly then a defined result will occur. If the result is not obtained something went wrong with the experimental set up, stock regents or personal technique.

A PCR product obtained in a PCR may not always be the desired product. The main concern in PCR is contamination of the reaction with foreign DNA. The negative control contains all the reagents as your PCR amplification without the template DNA. If there is no template DNA to copy from, you should not see a PCR product.

Remember: The next step is to come into the lab to practice with the materials, view a demonstration, or to even just ask questions. Also, if you are ready to take the lab final, please inform your instructor. The lab final will be performed by yourself and will be graded with a rubric.