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/transformatio
n1.html
Purpose of this week’s lab
1. Splice genes for ampicillin and kanamycin resistance
into a recombinant plasmid
2. Transform E. coli bacteria with the recombinant
plasmid
3. 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
Understanding Sticky Ends
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