Report On Molecular Biology Workshop

Date:               1 August 2009

Time:               9.00a.m-4.00p.m

Venue:             INTI College University

Topic:              Restriction Enzymes & Agarose Gel Electrophoresis

Participants : Students of 6BS3, 6BS2 and 6AS3 led by Puan Sang Fay Lee

Students were divided into two groups and led by Mr Wong and Miss Choo      

Activities

1. Introduction to the types of micropipettes and the usage of  micropipettes to measure micro liter of substances.  

2. Introduction to the types of restriction enzymes and the mechanisms- The restriction enzyme used were ECORI and HINDIII. Brief explanation on blunt end and sticky ends.

3. Preparing the Restriction enzyme digest

   1. Restriction endonuclease Digestion Of Lambda phage DNA

-prepare three sterile 1.5-mL micro centrifuge tubes labeled “1” , “2” and “3” .

-add the reagents into the sterile 1.5-mL micro centrifuge tubes in the     

  order as listed.

-mix well and spin in the ultracentifugator with all the tubes in correct 

 order and add in a balancer to stabile the machine before spinning.

4. Explanation on how to predict restriction sites through on-line DNA analysis software. We use the NEBcutter.

   1. Retrieving Sequences of Internet ( http://www.ncbi.nlm.nih.gov/)

   2. Browse the Restriction Enzyme Analysis through internet

      (http://tools.neb.com/NEBcutter2/index.php)

c.   check the types and result of the lambda phage DNA finger printing

5. Steps to prepare agarose gel electrophoresis

   1. Preparing the gel

      -weight 0.6g of agarose into 250-mL culture buffer

      -transfer 40mL of electrophoresis buffer into the culture bottle  

        containing the agarose

      -microwave the agarose at relatively high heat for about 2 min until

        the powder is completely dissolved and the solution looks clear

      -place both rubber coasting dams and place the tray in the chamber

      -place the comb holder with the comb onto the tray

      -pour the molten agarose solution onto the center of the gel tray

      -cool and solidify the gel completely

      -once the gel has solidified, the comb and rubber end blocks can be         

        removed.

      -fill the chamber with electrophoresis buffer

2. Loading the gel

      -set the power supply at 200 V and turn it on for 5 minutes to “stack”

        the DNA against the wall of the gel well, then reduce to 75 V for the      

        remaining gel to run.

3. Staining the gel

-pour just enough 0.025% (w/v) methylene blue solution into the staining tray so that it cover the gel completely.

-dicard the methylene blue solution and replaced with several changes of water bath.

4. Determination of DNA restriction fragment size. Check the results and compare the DNA finger printing

5. All the groups have to present their results and compare the results by measuring the DNA fingerprinting and plotting a graph. It’s very time consuming because we have to produce the correct shape of the graph.

6. This has been considered as a colloquium for  form 6.

Closure

The workshop ended at about 4.00pm with Pn Sang presenting souvenirs to instructors and some photo sessions. We presented our colloquium by displaying our graph analysis and the finger printing banding. We were very happy because we gained a lot of knowledge during the workshop. We would like to express thousands of thanks to the instructors from INTI College University and our teacher Pn Sang Fay Lee. Inti College University is footing and fully sponsored the whole workshop including t transport and food. We were brought to tour the mini zoo where we were introduced to many rare species of insects,  lizards and turtles before we returned to our homes.