Protocols for ET recombination.

 

Protocols for ET recombination.

Oligo design

PCR reaction and recipient plasmid DNA preparation

Preparation of competent bacterial cells

Transformation

Using Cre or Flp transient plasmids

Using Cre or Flp expressing E.coli

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).
   • 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.
   • 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.
   • 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.
   • 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.
   • Purify the PCR products by Qiagen column and elute with 2x 50 ul dH2O.
   • Add 10x Dpn 1 buffer and 2 ul Dpn 1 (NEB) and digest (to eliminate template DNA) for 1 hour.
   • 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)
   •  If competent cells harbour recipient plasmid, 0.3 ug of PCR product will be used for transformation.
   • 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.
   • Transform your host with the ET expression plasmid you are using by standard procedures and plate.
   • Pick single colony and grow in 5 ml LB medium overnight.
   • Transfer 0.7ml into 70 ml of LB medium (without glucose!) and grow them at 37oC with shaking.
   • Prepare 10% glycerol with dH2O, and cool down on ice for at least 3 hours before using.
   • When the cells reach OD600 = 0.1-0,15, add 0.7ml 10% L-arabinose to induce ET protein expression.
   • After a further 45-60 minutes, the cells should be at OD600 of 0.3-0.4. Harvest.
   • make sure the centrifuge and SA600 or SS34 (Sorvall) rotor is very cold by centrifuging for 10 min, -5oC at 4,000 rpm.
   • Spin 35 mls cells for 10 min at 7,000 rpm at -5oC. Put the other 35 mls on ice.
   • Pour away the supernatant, add the second 35 mls and respin.
   • Pour away supernatant, put tube on ice, resuspend cells in 5 ml ice cold 10% glycerol with an ice cold 5ml pipette. Add a further 25 mls and centrigue.
   • Repeat the above step twice.
   • Pour away supernatant and immediately dry the tube out with Kleenex tissue takning care not to touch the pellet.
   • Resuspend the cells in the remaining liquid (you should have a little more than 100ul final resuspended volume).
   • Transfer 50 ul of cells into each pre-cooled eppendorf tube and freeze in liquid N2 or use immediately.
     All the steps should be done as cold as possible, always on ice! The glass pipettes should be cooled once or twice by pipetting cold 10% glycerol up and down before pipetting cells.
   • (If cuvettes were reused) Wash cuvettes at least 10 times with dH2O, precool them on ice for least 5 min.
   • Thaw competent cells on ice and add 1 ul of PCR product (for transformation) or 1 ul mixture of PCR plus DNA for co-transformation.
   • Electroporate the cells at 2.3 kV (Bio-Rad Gene Pulser), 25 uF with Pulse controller set to 200 ohms)
   • Add 1 ml of LB medium and transfer back into the eppendorf tube.
   • Incubate at 37oC for 1 to 1.5 hours with shaking.
   • Plate 100ul on suitable antibiotoc plates, spin the rest, resuspend in 100ul and plate that..
705-Cre and 705-Flp plasmids are based on the pSC101 temperature sensitive origin. This origin maintains a low copy number and replicates at 30oC. These plasmids will be lost from cells when they are incubated at temperatures above 37oC. In addition, Cre and Flp are expressed from the lambdaPR promoter, which expresses weakly at 30oC and strongly at 37oC. Thus 705-Cre and 705-Flp can be used to give a transient burst of expression after which they will be eliminated so the recombined product can be isolated uncontaminated by their presence.
2. • Transform the E.coli host containing the plasmid to be site specifically recombined with 705-Flp or 705-Cre. (The method of transformation is not critical, we use RbCl2 competent cells because it is simpler than preparing electrocompetent cells).
   • Incubate at 30oC for 1.5 hr with shaking after transformation.
   • Plate on L.B. plates containing 25 ug/ml of chloramphenicol (Cm).
   • Incubate at 30oC for 2 days.
   • Pick single colonies and grow a mini-prep in L.B. medium overnight at 37-38oC (not 35oC!).
   • Streak onto L.B. agar plate without chloramphenicol, and culture at 37oC. (During incubation at 37oC, Flp or Cre will be expressed and recombine FRT or loxP sites. At the same time, the 705 plasmid will be lost.)
   • Pick single colonies and streak on Cm plates to check if the 705 plasmid is lost.
294-Cre and 294-Flp were generated by integrating 705-Cre and 705-Flp into the lacZ locus of E.coli strain, MM294 (Buchholz et al, Nucleic Acids Res. 24, 3118-9 (1996).
3. • Transform the plasmid carrying loxP or FRT sites into 294-Cre or 294-Flp competent cells.
   • Incubate at 37oC for one hour.
   • Plate on the selection plates and incubate at 37oC over night.
   • Pick colonies from the plates and inoculate 5ml LB medium with appropriate antibiotics.
   • Incubate at 30oC over night. (The temperature should be 30oC so that Cre or Flp expression is reduced. We often observe that the yield of plasmid DNA from cells strongly expressing Cre or Flp can be very low.)
   • Check the plasmid by mini-prep to see the loxP or FRT sites lost or not.