BISC411
EXPERIMENTAL MOLECULAR BIOLOGY OF THE CELL
BATCH INDUCTION PROTOCOL
1. In late afternoon of the previous day, instructor inoculates two bacterial cultures, one with the lysozyme expressing plasmid and another with the expression plasmid you were given in week 2. You will know the identification of this second plasmid now, determined in week 5. The inoculation tubes each contain 10 ml of LB plus 100 ug/ml ampicillin. Instructor grows them overnight, shaking at 300 rpm at 37oC.
2. Approximately 3 hours before the start of the lab (around
9:30),
instructor
seeds 2 80 ml of LB containing 100 ug/ml
ampicillin with .8 ml of these overnight cultures (one for each
culture). Cultures are shaken at 300
rpm for about 3 hours. (To OD 600 of about 0.5)
3. Students remove 250 microliters of each culture and puts
each into a
clean microfuge tube labeled with the name of the culture.
4. Instructor makes fresh IPTG stock at 0.1 M just before
needed. (120
mg in 5 ml of water).
5. Students add 0.24 ml (240 microliters) of the IPTG stock to
each of the remaining cultures. (This is a total of .3mM IPTG).
Cultures are returned to shaker for 2 hours of induction.
6. Students spin down their 250 microliter of culture that
were removed above in step 3, decant, and
resuspend the pellets in 50 microliters of 2XEB (protein gel loading
buffer). Freeze tubes at -20oC. These are your uninduced samples for
later gel analysis.
7. During the remainder of the 2 hour shaking period students will give their oral presentations (see general schedule).
8. When the induction period is over, remove 0.5 ml of each
culture
into a clean microfuge tube, spin down, decant, and resuspend pellets
in 50 microliters of 2XEB . Freeze at -20oC. These are your induced but
not purified samples.
9. Students then divide the remaining culture from each sample into two 50 ml centrifuge tubes (total of four tubes per group, 2 for each culture), cap the tubes, and spin them in the large centrifuge at 4000 rpm for 5 minutes. We must share the centrifuge!!
10. Decant the sup. Resuspend each pellet in 3 ml of dialysis phosphate buffer. Combine the suspensions into one of the 50 ml tubes (Do not mix up the two cultures). Sonicate each in ice-slurry. We will do 3 20 second pulses at setting 8. Immediately put the sonicate tube on ice (never let it get warm).
11. Divide each sonicate into 6 microfuge tubes. Spin in the
cold room
(016
McKinly) microfuge for 5 minutes at 12000 rpm. Patience!! We must share
the microfuges.
12. Collect all supernatants from each culture together into a 15 ml conical tube. (total of 2 tubes). Keep cold.
13. Obtain 2 pieces of dialysis tubing and securely clamp one
end.
Put
the supernatants into the tubing using a pasteur pipette and taking
care
not to puncture the tubing.
Squeeze out most of the air above the sample in the tubing and clamp
that
end securely. Put the two bags into the beaker you were given.
14. Go to the cold room and add the dialysis buffer ( 20mM
sodium
phosphate,
pH 7.2) to fill the beaker The dialysis tubing with your samples should
be covered completely. Be sure they are
submerged. Use the flat end of a pasteur pipette to weigh them down if
needed. Label
the beaker with your section and group number.
15. Change the dialysis buffer 3 times by pouring the buffer into the 016 McKinly sink and refilling the flask with fresh buffer (from the cold room). Do this as follows:
First change, morning of the next day.
Second change, later that day. (Have at least 3
hours of time between the first and second changes).
Third change, anytime the day after that.
If you do not do these buffer changes, your sample will not be
completely
equilibrated for use in the protein purification protocol. The prep
room
is open during normal daytime hours (8:30 through 4:30).
Determine who in your group is responsible for each of these buffer
changes.