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° C 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° C 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° C 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.
上一篇：An AFLP-based genome-wide mapping strategy（基于AFLP技术的全基因组扩增策略） 下一篇：Qiagen MIDI protocol
