PROTOCOLS FOR INTRODUCTORY LAB (Lab 1)
Goals: To complete a restriction enzyme digest, PCR amplification, agarose gel and gel documentation in 4 hours.
PCR amplification
To perform a PCR amplification you require a template DNA, two primers, buffer, MgCl2, dNTPs and thermostable Taq DNA polymerase.
On your rack you will be supplied with all these components plus 2 PCR tubes.
Set up the following two reactions:
| component | Reaction 1 | Reaction 2 |
| template DNA | 1ul | 1ul |
| primer 1 | 1ul | 1ul |
| primer 2 | - | 1ul |
| dNTP | 2ul | 2ul |
| 10X taq buffer | 5ul | 5ul |
| 50mM MgCl2 | 3ul | 3ul |
| water | 37.8ul | 36.8ul |
| Taq DNA polymerase | 0.2ul | 0.2ul |
| Total volume | 50ul | 50ul |
Make a Master Mix that contains the dNTPs, Taq buffer MgCl2 the correct amount of water for reaction 2. Add 1ul of water to reaction 1 to give the correct volume. Pipet some of the Master mix into the tube containing the Taq polymerase and mix well. Take the well mixed enzyme solution and mix it with the rest of the Master mix. Add the master mix to the template DNA primer1 and primer 2. Give the reactions to the TA to place in the PCR machine.
Ten cycles 94° for 30sec--denaturation; 55° for 30 sec--hybridization and 73° for 45sec--extension.
When the PCR is complete remove 20ul of the reaction to a 1.5 ml microcentrifuge tube and add 10ul of sample loading buffer.
Restriction enzyme digestion
To perform a restriction enzyme digestion you require DNA to digest, buffer, and restriction enzymes. On your rack you will be supplied with all these components.
Set up the following 5 reactions in 1.5 ml microcentrifuge tubes.
| 1 | 2 | 3 | 4 | 5 | |
| DNA | 2ul | 2ul | 2ul | 2ul | 2ul |
| 10X buffer | 2ul | 2ul | 2ul | 2ul | 2ul |
| EcoRI | 0.5ul | 0.5ul | 0.5ul | ||
| BamH1 | 0.5ul | 0.5ul | |||
| HindIII | 0.5ul | 0.5ul | |||
| water | 15.5ul | 15.5ul | 15.5ul | 15ul | 15ul |
| total | 20ul | 20ul | 20ul | 20ul | 20ul |
Place in 37°C waterbath for 1 hour. Add 10ul of sample loading buffer.
You are going to load an 8 lane agarose gel. For a description of pouring and running an agarose gel see Agarose Gel .
You will then take a picture of the gel see Gel documentation.
You are to arrange your lanes in the following order: Lane 1 will contain 1ul of ladder added to 30ul of sample buffer (this may have been made up for you check with TA). Lane 2-6 will be the restriction enzyme digests. Lanes 7 and 8 will be the PCR reactions.
REMEMBER TO SAVE YOU IMAGE AS A .TIF FILE (RAW DATA). AND MAKE SURE YOU HAVE SAVED IT TO THE RIGHT FOLDER.
The schedule for this lab session is as follows:
0-30 minutes: PCR reaction. Start PCR machine at 30 minutes
30-60 minutes: Restriction enzyme digests. Have the reactions in the water bath by 60 minutes.
1hour-1hour 20 minutes: Tape and pour 1% agarose gel
1hour 50 minutes-2 hours: Add sample buffer and load the agarose gel.
2-3 hours: Run the gel. Gel documentation demonstration.
3 hours- 4hours: Stain the gel and record the image.
Marking scheme for the results of the introlab. Total 2.5 marks
Lane 1 ladder 0.5 marks
Lanes 2-6 0.1 mark per lane for presence of DNA. 0.1 mark per lane for restriction enzyme digested DNA.
Lanes 7-8 1 mark for no product in the control lane and a PCR product of the correct size in lane 8