Single Clone Excision Protocol

1.Pick plaques and put in 500 ml sterile SM with 20 ml chloroform.Store in 4° overnight.

2.Be sure we have SOLR and XL1-Blue cells in 10 mM MgSO₄,no more than 1 week old.If not,set up a culture of each in LB supplemented with maltose and MgSO₄ (add 500ml 20% maltose and 500ml 1 M MgSO₄ to 50 ml LB broth).

Next day:

3.If you're making new cells,spin them down 5 min at 1500 x g in the C0650 rotor in the Beckman centrifuge,then resuspend in about 40 ml 10 mM MgSO₄,and quantify absorbance.

4.In a 15 ml conical tube,place

200 ml XL1-Blue MRF' cells (at A600­­ of 1.0; if absorbance differs,adjust amount proportionally)

250 ml of phage stock (from step 1 above.Make sure that you don't transfer chloroform from the bottom of the tube).

1 ml ExAssist phage (it's in the -80,second shelf from bottom,in a zip-lock bag with several microfuge tubes)

5.Incubate tube at 37° for 15 minutes.

6.Add 3 ml LB broth and incubate 2.5-3hours at 37° with shaking (loosen lids and tape them down with masking tape so they'll be aerated).You can let these go overnight if you want.(possible stopping point)

7.Heat the tube to 65-70℃ for 20 minutes,then centrifuge at 1000xg for 15 minutes.

8.Decant supernatant into a new sterile 15 ml conical tube.This is a stock of excised pBluescript plasmid packaged in phage protein coats.You can keep this at 4℃ for a couple of months.(possible stopping point)

9.Put 200 ml of SOLR cells from step 3 (or adjust volume proportional to absorbance)to each of two microfuge tubes.Add 10 ml of phage supernatant to one,and 1 ml to the other (up to 100ml may be used if the excision hasn't gone well,but usually 1 ml is enough.)

10.Incubate tubes at 37℃ for 15 minutes.

11.Normally,the titer of transfected cells is so high that you can just take a loop full of cell suspension from each tube and make a streak plate.If the excision hasn't gone well,you can plate 200 ml of the cell mixture from each tube onto LB-amp agar plates.In any case,use LB-amp agar (SOLR cells aren't amp resistant,so this selects for the transfected cells).If you want to do blue-white screening,spread 100 ml each of X-Gal (2% in dimethylformamide,stored in 15 ml conical tube in 4°)and IPTG (40mM in dH₂O,stored same way)on the plate 30 min before plating.