Reference no: EM133540408

Molecular and Cell Biology

Practical - Cloning of a gene sequence coding for a protein of interest by PCR

Learning Outcome 1: Appreciate PCR is a powerful technique to amplify DNA sequence and in gene cloning
Learning Outcome 2: Understand how DNA sequences can be amplified by PCR
Learning Outcome 3: Understand how primers determine the region which is amplified
Learning Outcome 4: Recognise the function of temperature in PCR
Learning Outcome 5: Be able to set up and analyse a PCR experiment
Learning Outcome 6: Understand the steps involved in cloning by PCR

Introduction and objectives

You will perform a PCR using primers specifically designed to amplify the gene of interest and analyse your results by gel electrophoresis. You will observe the effect of different temperatures on PCR specificity using the temperature gradient mode of the thermal cycler. You will schematically describe the whole cloning process, from PCR to selection of your transformants on LB agar + Ampicillin plates using a diagram (see genomic lectures for examples of diagrams).

Experimental Protocol

You will obtain a DNA sample
You will amplify the whole gene in the DNA sample by PCR.

Materials
• Wear gloves and use sterile tubes and tips
• Thermal Cycler
• DNA template
• 2 X PCR Premix (with DNA polymerase, dNTPs, buffer and MgCl2)
• Forward PCR primer (10 µM)
• Reverse PCR primer (10 µM)
• Agarose

• TAE buffer
• 6X loading dye
• Molecular Weight Marker (100 bp DNA ladder or as indicated)
• Your PCR products

Methods

1. Calculate the volumes required for your 20 µl reactions. Refer to Table below.
• Template is supplied at X ng µl-1
• You need to allow for 2 µl of template per reaction.

• You will be supplied with three 0.2 ml reaction tubes which you will label 1 to 3.

2. Add all components to the bottom of each tube.
• You need to add 2 µl of template DNA to tubes 2 and 3, and 2 µl of water to tube 1 (negative control) to the very bottom of the reaction tube.
• Add all remaining components as calculated to the bottom of each tube.

3. PCR tubes will be loaded in to PCR cycle
• Initial denaturation
• 30 cycles of:
• denaturation
• annealing
• extension
• Final extension
*** PCR conditions (time and temperature of cycles) will be provided during class***

4. During the PCR prepare a 2% agarose gel with 1X TAE.

5. Load all PCR samples and DNA standard and run the agarose gel electrophoresis (2% agarose gel at 100 V for 30 min or until the orange dye is at the very bottom of the gel).

6. Document your results on the transilluminator and obtain a gel image.

7. Analysis of Results
Additionally to your PCR results you will be provided with a temperature gradient PCR gel image obtained on the gene of interest with the provided range of annealing temperatures. You will have to discuss what you observe in your report

Report:
Aim (10%): Concise statement about what we aimed to achieve today. This should include the overarching aim of this report and the specific aims

Place photographs of your gel as well as the one provided for the gradient experiment in the results section (clearly annotated)
• Include a calculation section showing how you worked out your values
• Describe your primer sequences in 5'-3' direction and calculate their Tm. Underline restriction sites used for cloning in their sequences. Start and stop sequences must be shown in bold
• Tabulate your migration data using a standard curve and include your curve in the results.
• Compare the mobility of the PCR products with the size markers and its expected size.
• Estimate the approximate sizes of each of your bands.
• Did you observe any bands in your no DNA control?
Concluding remarks/Discussion (40%): (~300-400 words)
Discuss the effect of different temperatures on PCR and how you will clone your PCR product.
• Discuss how you chose your primers and their Tm

• Discuss the effect of different temperatures on PCR specificity.
• Describe schematically the whole cloning process into the provided vector, from PCR to selection of your transformants on selective LB agar + antibiotic plates using your own diagram produced with powerpoint or equivalent software (see genomic lectures for examples of diagrams).