Exercise Exercise Exercise Exercise 6 66 6

TA Cloning TA Cloning TA Cloning TA Cloning

Introduction Introduction Introduction Introduction
Terminal A (TA) cloning is a method of cloning PCR products without the use of
restriction enzyme digestion. The method makes use of the template-independent addition of
an adenine on the 3 end of PCR amplicons by Taq polymerase. Corresponding linearized
plasmids with T overhangs are ligated to the PCR products, as they each have complementary
single nucleotide ends.
In this activity, we will employ TA cloning on the MAD20 strain P.falciparum MSP1 block
2 protein, and compare the results with sticky end ligation in subsequent experiments.

Materials Materials Materials Materials

Reagents
TA cloning kit
o Salt solution
o Linear vector
freshly-produced PCR amplicon of gene of interest
nuclease free water

Equipment
Thermocycler
Laminar flow hood
Shaking incubator set to 37C
Blue and white pipettors

AdditionalMaterials
LB-ampicillin agar plates
PCR tubes

Procedure Procedure Procedure Procedure

1. Amplify gene of interest using the appropriate conditions.
2. Set up the TA cloning reaction in a 0.2ml tube as follows (*keep all reagents on ice. store all
reagents, except salt solution and water, at -20C when finished).

Reagent* Reagent* Reagent* Reagent* V VV Volume olume olume olume
Fresh PCR product 1.5 uL
Salt Solution 0.5 uL
sdd Water 0.5 uL
Vector 0.5 uL

3. Mix reaction gently and incubate for 30 seconds to 30 minutes at room temperature (22-
23C).
4. Transform all of the TA cloning mixture into chemically-competent E.coli using heat-shock
transformation (refer to Exercise 5). Plate 20 uL, 50 uL and 100uL aliquots onto LB-amp
plates

Guide Questions Guide Questions Guide Questions Guide Questions to be given with Exercise 8 to be given with Exercise 8 to be given with Exercise 8 to be given with Exercise 8