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From 2010.igem.org

Fusion PCR

Annealing temperature=54 C

Fusion Construct 1

DNA Templates(Fusion primer amplified):

E1 - 1uL

Term1 - 5.05uL

J23106 - 5.05uL

E2 - 1uL

E3 - 1uL

Term2 - 5.05uL

10xBuffer- 2.5uL

ddH20 - 0.5uL

Fwd primer- 0.2uL

Rvs primer- 0.2uL

Taq - 0.2uL

Fusion Construct 2

DNA Templates (Fusion primer amplified):

E4 - 1uL

E5a - 1uL

E5b - 1uL

Term - 5.15uL

10xBuffer- 2.5uL

ddH20 - 10uL

Fwd primer- 0.2uL

Rvs primer- 0.2uL

Taq - 0.2uL

Meanwhile:

8 stock solutions are being made overnight--> 8 colonies with Amp resistance

E5a x 1

E4 x 2

E2 x 2

E3 x 2

E1 x 1

Gel Extraction--> Terminator (F1 & F2 didn't work)

0.19g of gel = 0.19mL gel

Gel : L3 Buffer

1 : 3

0.19mL: x

x=0.57mL

[Terminator 2]=21.4 ng/uL

PCR Fusion

F1: E1-T1, T1-E1, P1-E2, E2-E3, E3-T2

F2: E4-E5a, E5a-E5b, E5b-T

added 1uL of Enzyme DNA

added 3uL of Terminator (T) and Promoter (P) DNA