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ADE 2 PCR protocol

Round 1 PCR

In the first round, the kanMX4 module is amplified using the TAG 56mer and the DOWNSTREAM 64mer as primers. Resuspend the stock primers to a final concentration of 10uM. The final concentrations/amounts are shown in parentheses.

5ul 10x Taq buffer (see below)
0.5ul 20mM dNTP's (.2 mM)
0.5ul 5 U/ul Taq Polymerase (2.5 units)
5ul pFA-kanMX4 plasmid (~30 ng)
5ul 10uM ADE2 TAG 56mer (1uM)
5ul 10uM ADE2 DOWNSTREAM 64mer (1uM)
29ul water

50 ul final volume

*10x Taq buffer contains: 100 mM Tris-HCl (pH 8.4), 500 mM KCl, 15 mM MgCl2.


*The Taq Polymerase should be added last and PCR mixture should be kept on ice until the PCR is started.

PCR conditions (Perkin Elmer 9600):
3 min, 94 °C (initial denaturation)
15 sec, 94 °C --->
15 sec, 54 °C ---> 25 cycles:
60 sec, 72 °C --->
3 min, 94 °C (final elongation)


Load 2ul on a 2% agarose TAE gel



The predicted 1.5 kb PCR product was obtained .


Round 2 PCR

In the second round, the PCR product from the first round is amplified using UPSTREAM 63mer and the DOWNSTREAM 64mer.

5ul 10x Taq buffer (see below)
0.5ul 20mM dNTP's (.2 mM)
0.5ul 5 U/ul Taq Polymerase (2.5 units)
2ul Round 1 PCR product
5ul 10uM ADE2 UPSTREAM 63mer (1uM)
5ul 10uM ADE2 DOWNSTREAM 64mer (1uM)
32ul water

50 ul final volume

*10x Taq buffer contains: 100 mM Tris-HCl (pH 8.4), 500 mM KCl, 15 mM MgCl2.

*The Taq Polymerase should be added last and PCR mixture should be kept on ice until the PCR is started.



PCR conditions (Perkin Elmer 9600):

3 min, 94 °C (initial denaturation)
15 sec, 94 °C --->
15 sec, 54 °C ---> 25 cycles:
60 sec, 72 °C --->
3 min, 94 °C (final elongation)


Load 2ul on a 2% agarose TAE gel


This PCR product can be directly used in the transformations.