Title: Screening Bacteria for Recombinant bglA-pLATE31 Plasmid
Course: BTEC-1100-301-302-303-F21 > Section: Module 4 - Applied Molecular Biology

You should now hopefully have many bacterial colonies growing on the LB agar/amp plate.  We will now screen several colonies to verify that our gene has been cloned into the pLATE plasmid.  To do this we will use PCR.  In the screening PCR we will use the identical primers that we used to initially amplify the bglA gene.  The only difference from the screening PCR and the one we completed in module 3 is the DNA template.  In this case we will be using colonies from the growth plate to serve as the template.  We want to screen 5 colonies from the growth plate so you will need to set up 5x50 μl  reactions.

Protocol for PCR

Stock Reagents for PCR:

- 12.5 μM forward primer (BGLALICFWD)
- 12.5 μM reverse primer (BGLALICREV)
- 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 (l) of Stock Solution Required to Make Reaction Mix
12.5 μM forward primer (BGLALICFWD) | 0.25 μM forward primer |
12.5 μM reverse primer (BGLALICREV) | 0.25 μM reverse primer |
10X PCR buffer | 1X PCR buffer |
25 mM MgCl2 | 1.5 mM MgCl2 |
2.5 mM dNTPs | 0.25 mM dNTPs |
1 unit/μl of Taq polymerase | 1 unit |
Bacterial colony | Bacterial colony |
water | — |
Total Volume | — | 50 μl

A positive  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.  The template for the positive control should be a bacterial colony of a strain already containing the bglA-pLATE31 recombinant plasmid.

Your instructor will demonstrate how to pick colonies and save them for future sequencing when you perform the lab.

Following the amplification you will run a gel to determine if your colonies contained the recombinant DNA.  This will be the same gel as we ran in module 3, a 0.8% agarose gel in 1X TAE.

Learning Objectives:

- Screen bacteria for the presence of the plasmid and gene using gel electrophoresis.