CBMS 336- Molecular Biology and Genomics

Requirements

? Students are required to satisfactorily complete all components of the unit to pass. Satisfactory completion of all components includes the practical assignments and the problem sets which are both integral components of the unit. Apart from the marks formally allocated to these components, a comprehensive understanding of them will greatly assist you in the final exam. You should remember that the final exam covers ALL components of the unit. 

Adhering to a few simple common sense rules will greatly assist in the running of the laboratory.

? We expect you to assist in the general running of the laboratory by cleaning your work area up before you leave. This includes items that you may have left in common areas.

? You are required to have a lab coat and should wear it at all times in the laboratory.

? You must wear appropriate closed-in footwear.

? Any contaminated glassware and pipettes should be immediately disposed of in the proper container. This includes any plastic disposable pipette tips.

? Spent liquid containing live microorganisms should not be tipped down the sink. Instead dispose of it in the labeled containers.

Any spilt cultures must be cleaned up immediately. Used agar plates should be placed in biohazard bags for autoclaving.

? Always wear gloves when handling compounds like phenol or chloroform in particular.

Many of the chemicals you will be using are toxic or carcinogenic or both. Dispose of tips contaminated with these compounds only in the containers labeled for the purpose.

? Always wear gloves when handling and loading gels.

? When looking at gels under ultra-violet light you must always wear the UV opaque glasses provided. Do not rely on normal eye wear for protection since such glasses do not provide protection at the side and are not necessarily UV opaque. As far as possible also minimise your exposure on the skin unless using block out cream.

Chemical and Expeerimental Risk Assesments

To avoid risk of injury from any of the chemicals or procedures used in these sessions it is mandatory to make a risk assessment. This has been undertaken on your behalf and the SDS (Safety Data Sheet) and risk assessment forms for both chemicals and activities will be available in the laboratory. It is essential that you read and familiarize yourself with these.

Step 1. Restriction digests and agarose gel electrophoresis

You are given DNA from the plasmid vector pTTQ18 and a PCR amplification product which

encodes a copy of either the amilCP or rfp gene.

1. Per pair of students set up restriction digests of each of pTTQ18 and the reporter gene PCR product (in separate tubes!) with the enzymes KpnI and SmaI. The reaction will be as

follows:

? 10µL DNA (pTTQ18 OR reporter gene PCR product)

? 6µL Sterile water

? 2µL 10x KpnI/SmaI Buffer

? 1µL KpnI

? 1µL SmaI

2. Put in microfuge tube rack on front bench to be incubated at 37°C for 1 hour.

3. Ray will do this for you: Load samples from the restriction digest and run on an agarose gel. Add 4?L 6? loading buffer to 20?L samples before loading the samples to the appropriate well. Make sure you know which sample is in which lane!

Note: The gel will contain 1.0% agarose and a small amount of GelRed (a non-toxic fluorescent dye used as a replacement for ethidium bromide, which is toxic) to allow visualization of the DNA under UV light after the run.

Note: We will load markers (a “DNA ladder”) on the gel so that the size of your bands can be estimated.

4. Run the gel for 30 min at 100 V. We will then demonstrate the visualization of the DNA using GelRed and how an image is taken of the gel using an imaging system.

5. Ray will gel purify your digested DNA from the agarose gel later in the week.

Step 2. Ligation

1. Per pair of students, use the restriction digested samples from last week to set up a ligation. We will use an insert to vector ratio of approximately 3:1.

2. Label your tubes carefully and place them in the lidded rack provided on the front bench. Fill out the record sheet provided with your details in the corresponding box.

3. Ray will incubate your samples at 16°C overnight, and then store them for the next week.

Step 3. Transformation

1. Per pair of students, take 5µL of your ligation mix and add to 50µL of competent E. coliJM101 cells. Mix and stand on ice for 15 minutes.

2. Heat shock cells at 42°C for 1 minute.

3. Add 950µL of LB, mix and incubate, shaking at 37°C for 40 minutes.

4. Plate 50µL of the sample onto Nutrient agar ampicillin plates.

5. Put in correct plate rack (please read the labels on the racks) to be incubated at 370C overnight.

Step 4.

Observe the colonies on the transformation plates, count blue/red and white colonies.

Step 5.

We have selected one example colony, and sequenced DNA isolated from the colony using primers pTTQ18_F and pTTQ18_R. You are provided with a chromatogram of the sequences for examination and discussion.