Easy Gene Splicer
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Transcript 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/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