catholic university of louvain
school of medicine
(brussels, belgium)
molecular genetics workshop, SBIM 2520

 université catholique de louvain
faculté de médecine
(bruxelles, belgique)
molecular genetics workshop, SBIM 2520
Jean-Pierre HERVEG herveg@bian.ucl.ac.be.nospam supress .nospam

first week of 1 and 29 July 02 workshops
FARM 2182, les phages et la PCR (Dr E De Plaen) and back to informations

tasks
phage lambda
----------------1. measure the pfu of a phage suspension
----------------2. prepare competent cells
----------------3. isolate lambda phage DNA (minipreps)

RT-PCR
----------------1. from dissection of the gland to extraction of thyroid total RNA
----------------2. RT PCR of ß-actin in thyroid RNA
----------------3. electrophoresis

background (in English or in French))
notions de génie
génétique pratique (farm 2182) par Etienne DE PLAEN PhD, phage lambda and PCR, endonucléase,
Gilson pipette,
recombinant lambda phage, a plaque, our spectrophotometer, measure DNA concentration, prokaryote incubator, eukaryote O2/CO2
incubator, RT-PCR theory, getting primers (an exercise), PCR: from the databank to the bench, program the thermocycler,
run the thermocycler, electrophoresis.


phage lambda 1st task:
measure the pfu of a phage suspension
pfu = plaque forming unit

1st task:
measure the pfu of a phage suspension

1. prepare 1/10 serial dilutions of a phage suspension of unknown title.
make a 1/10 serial dilutions as following:
in all tubes
add 270 µl of SM medium.
add 30 µl of phage suspension in the 1st tube
mix carefully
remove 30 µl from this mix, and add it to the 2nd tube
mix carefully
repeat this operation until the last tube.

2. infect a constant amount of E. coli with each phage dilution
transfer 100 µl of each tube of the upper row into the corresponding tube of the 2nd row.
add 100 µl of competent bacteria to each tube of the 2nd row
Incubate for 20 min at 37°C.

3. add 100 µl of an infected E. coli suspension to a glass tubes containing 3 ml of molten (47°C) top agar.
mix gently, avoiding air bubbles
Immediately pour the entire content of the tube on the center of a numbered petri dish, avoiding air bubbles.
swirl the plate gently to ensure an even distribution of bacteria in top agar

4. Incubate overnight at 37°C

5. the next day, count the pfu (plaque forming unit) of the original phage suspension.

go to plate


phage lambda 2nd task:
prepare competent cells (cell able to be infected by lambda phage)



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sterilize a capped tube containing 5 ml of LB medium.

let cool down to room temperature.

Introduce sterily the volume of maltose and MgSO4 you have computed.

Now introduce either a fresh MRAP II colony. You can also introduce 5 to 10 µl of MRAP II cells in a glycerol suspension (after, thawing the suspension!).

Incubate overnight at 37°C.

The next day, cells will express the lamb gene, a maltose receptor to which phage lambda is adsorbed before being internalized.

phage lambda 3rd task:
isolate lambda phage DNA from a bacterial lysate (minipreps)



 

 

 

 

 

 

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RT-PCR 1st task:
from thyroid dissection to RNA



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RT-PCR 2nd task:
RT- Polymerase chain reaction




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RT-PCR 3rd task:
electrophoresis to analyze the RT-PCR output



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first week of 1 and 29 July 02 workshops

 catholic university of louvain
school of medicine
(brussels, belgium)
molecular genetics workshop, SBIM 2520

 université catholique de louvain
faculté de médecine
(bruxelles, belgique)
molecular genetics workshop, SBIM 2520
Jean-Pierre HERVEG herveg@bian.ucl.ac.be.nospam supress .nospam
back to
informations