Splice Overlap Extension (SOE)
SOE utilizes the PCR thermal cycler to anneal two DNA fragments with partially overlapping
complementary 3’ ends
The polymerase reaction then extends the fragments, using the other annealed fragment as a template
This, in essence, creates a double stranded fragment the full length of the combine two starting pieces
The primers used allow further amplification of the new full length fragments after the initial extension
µL
F
R
TF
TR
5
2
42-(F+R+ TF+ TF)
1
50
Component
Forward Primer (5’)
Reverse Primer (5’)
Template Forward Fragment
Template Reverse Fragment
Pfu Buffer
dNTPs [10 mM]
H2O
Pfu Polymerase
Total
Notes
180 ng
180 ng
10X
To final
2.5 U/µL
Run using cycling protocol from Mutagenesis section, adjusting for extension times based on overall length
of final annealed products
Amounts of TF and TR to use vary based on concentration of starting product. Total mass should probably
be 75 to 150 ng