244
ANNETTE DEICHMANN AND KLAUS DEICHMANN
Standard reaction
3. To make the JM103 cells competent, follow the procedure described in
Subprotocol1, Procedure, steps 2-10. In place ofLB medium use 2 x YT
medium. In step 4 remove 2 ml of the log-phase cells and store them in a
sterile tube at 4 °C.
4. Mix 10-100 ng of recombinant M13 DNA with 200 J..Ll of competent
JM103 cells.
5. Place on ice for 40 min.
6. Heat shock the cells at 42°C for exactly 2 min. Place them on ice immediately.
7. For the following steps, sterile glass tubes, 2 x YT agar plates and 2 x YT
top-agar must be prewarmed at 48°C.
8. Divide the transformation mixture by two: To one prewarmed glass
tube add 20 J..Ll to another 180 J..Ll of the transformation mixture.
Keep the tubes at 48°C.
9. To each of the glass tubes add:
Log-phase JM103 cells from step 3
200 Jll
100 mM IPTG
10 Jll
2o/o X-gal solution
SO Jll
2 x YT top-agar
3 ml
10. Mix well and pour each tube immediately on to a 2 x YT agar plate
prewarmed at 48°C. Swirl the plate so that the surface of the 2 x YT
agar is covered.
11. Allow the top-agar to harden which takes approximately 15 min.
12. Incubate the plates inverted overnight at 37°C. Clear plaques correspond to transformed cells bearing an insert, blue plaques to those without insert.
13. It may be necessary to prepare 5-10 plates with IPTG, X-gal solution,
JM103 cells and top-agar to streak out the recombinant plaques the next
day.
ANNETTE DEICHMANN AND KLAUS DEICHMANN
Standard reaction
3. To make the JM103 cells competent, follow the procedure described in
Subprotocol1, Procedure, steps 2-10. In place ofLB medium use 2 x YT
medium. In step 4 remove 2 ml of the log-phase cells and store them in a
sterile tube at 4 °C.
4. Mix 10-100 ng of recombinant M13 DNA with 200 J..Ll of competent
JM103 cells.
5. Place on ice for 40 min.
6. Heat shock the cells at 42°C for exactly 2 min. Place them on ice immediately.
7. For the following steps, sterile glass tubes, 2 x YT agar plates and 2 x YT
top-agar must be prewarmed at 48°C.
8. Divide the transformation mixture by two: To one prewarmed glass
tube add 20 J..Ll to another 180 J..Ll of the transformation mixture.
Keep the tubes at 48°C.
9. To each of the glass tubes add:
Log-phase JM103 cells from step 3
200 Jll
100 mM IPTG
10 Jll
2o/o X-gal solution
SO Jll
2 x YT top-agar
3 ml
10. Mix well and pour each tube immediately on to a 2 x YT agar plate
prewarmed at 48°C. Swirl the plate so that the surface of the 2 x YT
agar is covered.
11. Allow the top-agar to harden which takes approximately 15 min.
12. Incubate the plates inverted overnight at 37°C. Clear plaques correspond to transformed cells bearing an insert, blue plaques to those without insert.
13. It may be necessary to prepare 5-10 plates with IPTG, X-gal solution,
JM103 cells and top-agar to streak out the recombinant plaques the next
day.