Easy Gene Splicer Carolina Biological Kit #21-1162
Presented by Bryan Smith
Background • In the field of biology, what is transformation? • What is a plasmid? • Why are plasmids used in biotechnology? • What properties should the plasmid have?
Background for this week’s lab • http://www.dnalc.org/ddnalc/resources/transformation1.html • Purpose of this week’s lab • Splice genes for ampicillin and kanamycin resistance into a recombinant plasmid • Transform E. coli bacteria with the recombinant plasmid • Isolate the transformed bacteria by growing them on plates with ampicillin and kanamycin
Day #1 • Procedure A – Set up Ligation reaction • Materials needed • pAMP digested by BamH1 & HindIII into two fragments: 3755bp w/ampicillin resistance gene & 784bp • pKAN digested by BamH1 & HindIII into two fragments: 1875bp w/kanamycin resistance gene & 2332bp • Tube of Ligation buffer/Ligase/ATP: the enzyme that will re-bond BamH1 sticky ends and HindlII sticky ends • 20uL micropipettes
Ligation possibilities • BamH1 sticky ends can only be ligated to other BamH1 sticky ends and the same is true for HindIII sticky ends. WHY? • What are the possible ligation outcomes of procedure A?
Day #2 • Procedure B –Transform cells with ligated DNA • Work in groups of 3 • Each group will prepare 2 tubes of cells • One w/pAMP/KAN (labeled +pAMP/KAN) • One w/o pAMP/KAN (labeled –pAMP/KAN) • Each tube • Kept ice cold • Heat shocked • Returned to ice cold • Incubated for hour in Luria Broth(LB)
Procedure C-Plate cells in growth media • Each group will label w/group name & date • One LB plate • Two LB/AMP/KAN plates • Half the groups will plate • 100uL -pAMP/KAN cells on LB & LB/AMP/KAN plate • 100uL +pAMP/KAN cells on LB/AMP/KAN plate • Other half will plate • 100uL -pAMP/KAN cells on LB/AMP/KAN plate • 100uL +pAMP/KAN cells on LB & LB/AMP/KAN plate • Spread cells with sterile inoculation loop • Incubate overnight at 37oC
Controls • What is the positive control & what will it tell us? • What is the negative control & what will it tell us? • The teacher will prepare two ligation controls • Cells transformed with pAMP/ligase • Cells transformed with pKAN/ligase • What will these controls tell us?
Day #3 • Examine plates • How do know if transformation took place? • Go over RESULTS & DISCUSSION questions in hand out