Cloning blunt ended PCR products
This procedure is for cloning PCR products that have been generated by a high fidelity Taq DNA
polymerase, which leaves no A at the end of the DNA.
We will clone PCR fragments in order to sequence them. These fragments have blunt ends.
This means that they are completely double-stranded. We will therefore clone them into a blunt-ended
plasmid. The system that we will use uses a plasmid called pCR4 blunt-TOPO. This plasmid has a site
for Topoisomerase I from Vaccinia virus. When Topo I cleaves the plasmid, it covalently binds to the 5’
ends of the DNA. When we add our PCR fragment, the Topo I reaction is forced towards forming a
dsDNA, hence inserting the PCR fragment into the plasmid.
After using the Topo I reaction, we insert the plasmid into E. coli cells. Any cells containing
plasmid without an insert will kill the cell due to the ccdB gene on the plasmid. If our DNA is inserted,
the ccdB gene is missing, so the plasmid with inserts grows. After obtaining colonies, we will grow
them up, purify plasmid DNA, and sequence the DNA.
Materials
Zero Blunt TOPO PCR Cloning Kit for Sequencing (Invitrogen)
37 C and 42 C water baths
LB plates containing 50 g/ml kanamycin or 50-100 g/ml ampicillin (3 per cloning reaction)
LB glycerol (LB media containing 15 % glycerol)
Procedures
Topoisomerase reaction
1) We will use the PCR products from the PCR experiment. Mix together the following into a
microfuge tube. You should make up one sample for each of your PCR reactions.
Reagent
Volume ( l)
PCR product
4
Salt solution
1
TOPO vector
1
Final volume
6
2) Gently mix the reaction and incubate them at room temperature for 5 minutes to 30 minutes*.
Cloning
3) Place the reaction on ice.
4) Add 2 l of the sample from step 1 to a vial of One Shot Chemically Competent E. coli cells and
gently mix them. DO NOT MIX WITH A PIPETTE as they are very fragile.
5) Incubate them on ice for 5 minutes to 30 minutes*.
* This means that the reaction takes about 5 minutes to complete, but after 30 minutes the results start to get worse.
Last printed on: July 24, 2012
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6) Heat-shock the cells for 30 seconds at 42 C without shaking, then immediately place them on ice.
7) Add 250 l of room temperature SOC medium.
8) Cap the tube and incubate at 37 C for one hour.
9) Spread ~90 l onto each of three LB plates containing ampicillin or kanamycin. Incubate the plates
at 37 C overnight.
10) The next day, look at the plates. Count the colonies. Label and seal the set of plates and store them
in the refrigerator.
Clone banks
11) To make a clone bank:
Materials: You will need one 96-well plate, sterile toothpicks, and LB-glycerol media
a) Obtain a 96-well plate. Add 200 l of LB-glycerol media containing 50 g per ml kanamycin or
50 g per ml ampicillin.
b) Use sterile toothpicks to pick one colony per well. Stab the colony with a toothpick and put it
into a well containing the media.
c) Let the toothpick sit in the well only for a few seconds, as the toothpick will absorb moisture.
Remove and discard the toothpicks.
d) Incubate the 96-well plates at 37 C overnight.
e) Store in the -70 C freezer.
Analysis
Count the number of colonies on the plates. If the transformation didn’t work, what could have gone
wrong?
Last printed on: July 24, 2012
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