Have performed PCR of Spycatcher gene (~339 bp) and Pal gene (~498 bp) with their respective primers, P1 forward and P2 reverse, and P3 forward and P4 reverse, respectively. Ran a 1% agarose gel and performed gel extraction and purification of each DNA. Did a check of the Spycatcher and Pal DNA, they were clean as single bands for each PCR product were observed from 1% agarose gel (size gauged by 100 bp ladder).

Next, performed overlap PCR to fuse Spycatcher and Pal. However, obtained multiple bands (600 bp to 1500 bp) after resolving in 1% agarose gel (size gauged by 100 bp ladder).

Had performed the reaction at annealing temperature of 58 deg C (trial #1), 2 uM primers (trial #2), gradient of annealing temperatures of 56 deg C, 60 deg C and 62 deg C (trial #3).

Note: Spycatcher and Pal gene had GGGGS tag being inserted, at the 3' end and 5' end respectively, in the 1st PCR run.

PCR set-up: 50 uL reaction

MilliQ water of 31.5 uL

5x Q5 Reaction Buffer of 10 uL

10 uM dNTPs of 2 uL

10 uM forward primer of 2 uL (P1 forward) [trial #1 & #3: 2 uL; trial#2: 0.4 uL]

10 uM reverse primer of 2 uL (P4 reverse) [trial #1 & #3: 2 uL; trial#2: 0.4 uL]

Template, total of 2 uL (1 uL of Spycatcher and Pal PCR product each, with concentration of 12 ng/uL and 16.4 ng/uL respectively)

Q5 polymerase of 0.5 uL

Thermocycling conditions:

Initial Denaturation, 95 deg C, 5 min

30 cycles:

Denaturation, 98 deg C, 10s

Annealing, 58 deg C, 30s (trial #1); gradient of 56 deg C, 60 deg C and 62 deg C (trial #3)

Extension, 72 deg C, 30s

Final extension, 72 deg C, 2 min

Hold at 4 deg C