Protocols for ET recombination

1、Oligo design

（1）The 5' end (the homology arm) - choose 42 or more (we usually choose about 50) nts for the homology arms from the target DNA sequence simply according to where you want to ｉｎｓｅｒｔ the PCR fragment. (For efficient oligonucleotide synthesis, the EMBL oligo service prefers that the 5' most nucleotide is a T or C. Consequently most of our oligos start with a T or C however we do not think this is important for ET cloning efficiencies).

（2）The 3' end (the PCR primer) - choose about 18-30 nt for the PCR primer region as you would for any other PCR. Use of the Oligo 4 software program can help, especially for shorter primers.

（3）Between these two regions, you can stick whatever you like - within the limits of oligo synthesis. We routinely put loxP or FRTs (each 34 bps) here, for subsequent recombination by Cre or FLP recombinases respectively.

2、PCR reaction and recipient plasmid DNA preparation

（1）The oligo powder (40 nmol without FPLC purification) was dissolved in 500 ul of dH2O. Extract oligo solution with phenol/chloroform as follows:

100 ul oligo solution

12 ul 3M NaAc (pH 7.5)

120 ul phenol:CHCl3

Vortex for 30', spin for 2-3 min, add 360 ul ethanol, place in -80oC freezer for 5-10 min, spin for 5 min, wash once with 70% ethanol, dry under vacuum and dissolve in 100 ul of dH2O.

（2）PCR reaction

33 ul dH2O

5 ul 10 x PCR reaction buffer

5 ul 2.5 mM dNTP (10 x)

1.5 ul upper oligo

1.5 ul lower oligo

2 ul templat

0.5 ul Taq polymerase (5 U/ul)

Annealing temperature is important, usually 60oC - 62oC is optimal.

（3）Purify the PCR products by Qiagen column and elute with 2x 50 ul dH2O.

（4）Add 10x Dpn 1 buffer and 2 ul Dpn 1 (NEB) and digest (to eliminate template DNA) for 1 hour.

（5）Extract with Phenol:CHCl3 once, precipitate with ethanol as above and redissolve in 5 ul dH2O (from 50 ul original PCR products, about 0.3 ug/ul)

（6）If competent cells harbour recipient plasmid, 0.3 ug of PCR product will be used for transformation.

（7）For co-transformation, extract recipient DNA once with phenol:CHCl3 as above and dissolve in dH2O at 1 ug/ul. Mix PCR products with recipient plasmid DNA (0.20-0.3 pmol PCR + 0.10-0.2 pmol vector per ul). 1 ul of mixture will be used for transformation.