basic introduction to genetic PGLO experiment

1. The Write upof the geneticengineeringpractical.
PGLO experiment.
In the PGLO experiment,the firstthingthatwasdone,wastomark the tubesbeingused,one witha
plussign (+),and the otherwitha minussign (-),sothat they can be toldapart.
The secondthingthat was done,wasthat the transformationsolutionwasaddedtothe tubes.The
thirdthingto be done was the tubesbeingputinice,to cool downthe bacteriaso that the heat
shockis more effectiveonthe bacteria.
The nextthingthatwas done wasthe introductionof the bacteriato the transformationsolution,
withthe use of a sterile loop.
The fifththingto be done wasthe examinationof the plasmidDNA withaUV lamp (UV Light) to
make sure that it iswhat we are after,the byusinga sterile looptake some of the plasmidand
introduce itto the +pGLO tube.
The sixthstepwasto incubate the tubesinthe ice for 10 minutes,take care to make sure that the
basesof the tubesare incontact withice.
The nextthingto be done was the labellingof the agar plates,twomarkwith+ and two markedwith
-,the two+ platesshouldbe labelledLB/ampandLB/amp/ara,whilstthe - platesshouldbe labelled
LB/amp andLB.
The eighthstepwasthe heatshockitself,inwhichthe tubescontainingthe bacteriaare both
transferredtothe waterbath, whichissetat 42o
C, theyshouldbe keptinthe bath for50 seconds,
againmake sure the bottomof the tubeshave contactwiththe warm water,afterthe 50 seconds
are up,put the tubesback onice quickly,andleave there for2minutes.
The ninththingto be done,wasthe removal of the tubesfromthe ice,inwhichthere shouldbe left
on the table top, usingtwosterile pipettes(oneforeachtube) add250 µl of LB broth to eachtube.
Incubate the tubesfor10 minutesatroom temperature.
The tenthstepthat happenedwasthe mixingof the solutionsinthe tubes,byclosingthemand
tappingthemwitha finger,the mixingoccurs.Usingseparate pipettesforeachplate add100 µl of
the solutionstothe appropriate plates.
The secondto last stepwasthe spreadingof the solutionoverthe plates,bythe use of sterile loops.
Thiswas achievedbyquickly“skating”the flatside of the loopacrossthe agar.
The last stepwasthe stackingandpackingof the plates,inwhichthe plateswere stackedontopof
one anotherand tapedto staytogether,the stackwas thenlabelledwiththe groupname chosenby
the individualspartakinginthe experiment.