342
MARTIN MARX
Procedure
1. Prepare five 15 ml Falcon tubes with 50 J..Ll competent HB101 (or any
other suitable bacteria). See Chapter 15 for the preparation of competent bacteria.
2. Add 20 J..Ll of the ligation mixture A, B, C, D or E.
3. Incubate 30 minutes on ice.
4. Heat shock at 42°C for two minutes.
5. Add 500 J..Ll LB media (see Subprotocol1) into each tube.
6. Shake the tubes at 250 rpm at 37°C for 30 minutes.
7. Plate the bacteria on 100 mm petri dishes with LB agar supplemented
with 100 J..Lg/ml ampicillin (see Subprotocol1, Materials for preparation
of LB agar). Set up four dishes with the ligation mixtures A, B and C
each, one dish with the mixture D ("positive control") and E ("negative
control")
8. Incubate the petri dishes overnight at 37°C.
9. The number of colanies on the plate should not decrease with increasing insert/vector ratio. The positive control D (vector alone) will probablyrender the highest number of colonies, whereas the negative control E (insert alone) should allow onlythe growth of a few, or ideally, no
colonies.
10. Pick a suitable number (usually 3-5) colanies from each ofthe plates A,
Band C, pick two or three colanies from plate D (vector alone). Transfer
each colony to a master plate with LB agar and 100 J..Lg/ml ampicillin
(tauch the plate first) and then, with a "second tauch", to a 15 ml Falcon
tube with 5 ml LB media supplemented with 50 J..Lg/ml ampicillin.
11. Incubate the master plates at 37°C overnight, then store at 4°C for possible future use.
12. Grow up the bacteria in the Falcon tubes in an orbital shaker (250 rpm)
at 37°C overnight.
13. Isolate the plasmid DNA bythe "miniprep" method (see Subprotocol4)
followed by phenol/chloroform extraction and ethanol precipitation.
14. Resuspend the precipitated DNA in 60 J..Ll of TE and store it at -70°C.
MARTIN MARX
Procedure
1. Prepare five 15 ml Falcon tubes with 50 J..Ll competent HB101 (or any
other suitable bacteria). See Chapter 15 for the preparation of competent bacteria.
2. Add 20 J..Ll of the ligation mixture A, B, C, D or E.
3. Incubate 30 minutes on ice.
4. Heat shock at 42°C for two minutes.
5. Add 500 J..Ll LB media (see Subprotocol1) into each tube.
6. Shake the tubes at 250 rpm at 37°C for 30 minutes.
7. Plate the bacteria on 100 mm petri dishes with LB agar supplemented
with 100 J..Lg/ml ampicillin (see Subprotocol1, Materials for preparation
of LB agar). Set up four dishes with the ligation mixtures A, B and C
each, one dish with the mixture D ("positive control") and E ("negative
control")
8. Incubate the petri dishes overnight at 37°C.
9. The number of colanies on the plate should not decrease with increasing insert/vector ratio. The positive control D (vector alone) will probablyrender the highest number of colonies, whereas the negative control E (insert alone) should allow onlythe growth of a few, or ideally, no
colonies.
10. Pick a suitable number (usually 3-5) colanies from each ofthe plates A,
Band C, pick two or three colanies from plate D (vector alone). Transfer
each colony to a master plate with LB agar and 100 J..Lg/ml ampicillin
(tauch the plate first) and then, with a "second tauch", to a 15 ml Falcon
tube with 5 ml LB media supplemented with 50 J..Lg/ml ampicillin.
11. Incubate the master plates at 37°C overnight, then store at 4°C for possible future use.
12. Grow up the bacteria in the Falcon tubes in an orbital shaker (250 rpm)
at 37°C overnight.
13. Isolate the plasmid DNA bythe "miniprep" method (see Subprotocol4)
followed by phenol/chloroform extraction and ethanol precipitation.
14. Resuspend the precipitated DNA in 60 J..Ll of TE and store it at -70°C.