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Inoculation of 7/20 Transformtion

Lots of colonies were present, 6 colonies were picked and inoculated with 5 mL LB broth and 5uL 1000X kanamycin.

Redo Transformation of psb1c3+T7 promoter+silk in BL21(DE3)

1. Thaw cells (~80uL) on ice for 10 minutes.
2. Add 1uL of ligated product.
3. Place on ice for 5 minutes.
4. Rescue in 320uL of SOC, incubate and shake for 1 hour.
5. Warm chloramphenicol plates at 37C.
6. Spin down bacteria to pellet, discard supernatant.
7. Add 111 uL of SOC, take 11uL and dilute 1:10 (11uL into 99uL).
8. Plate 100uL of bacteria (1:1 and 1:10) on each plate.
9. Spread with beads, incubate at 37C.

Inclusion Body Purification of Honeybee Protein

1. Determine wet wet weight of cell pellet after spinning liquid culter at 10000 x g for 10 min.
   1. 1.25 g
2. Resuspend in 5 ml/g Bug Buster (1x) by pipetting and gently vortexing.
   1. This is 6.25 ml in our case
3. Put on shaker or rotating mixer for 15 min at RT
   1. Took our first fraction at this point of the full cell lysate (F1)
4. Centrifuge 16000 g 20 min at 4 degrees C
   1. Took next fraction at this point of the supernatant, labeled (S1)
5. Resuspend pellet in same volume of 1X bugbuster as before
   1. 6.25 ml
6. Pipette up and down and vortex gently to get an even suspension.
   1. Took third fraction at this point (F2)
   2. I did not go to great lengths to get an even suspension here, but I noticed in retrospect that the protocol emphasized the importance of this in order to get pure inclusion bodies.
7. Add dry lysozyme to final concentration of 200 ug / ml
   1. For 6.25 ml solution I added around 1.25 mg of lysozyme
   2. Added 1 ul of DNAse to remove chromosomal DNA.
8. Add 6 volumes of 1:10 diluted bugbuster (.1X)
   1. At this point we split the solution up into two 50 ml falcon tubes and added 18.75 ml of .1X bugbuster to each tube
9. Centrifuge 16000g 15 min. 4 degrees C to collect inclusion bodies.
10. Remove supernatant w/ pipette, take next fraction (S3)
    1. After spinning this down, there was a pellet, with chromosomal DNA.
    2. It was difficult to remove the supernatant because the viscous liquid kept getting sucked up and bringing the pellet up with it.
11. Resuspend pellet in 1/2 volume of original 0.1X bug buster solution
    1. 9.375 ml per tube
12. Mix to get an even suspension by pipetting and vortexing for several minutes and spin down as in step 9.
13. Repeat was two more times.
    1. Take samples of supernatant at each wash step
14. Resuspend inclusion body pellet in 3% SDS solution and incubate at 60 C in the water bath for 2 hours
    1. I used around 2 ml, but I probably could have used less, because the pellet dissolves well in the heat
15. Store solution at 4C until further required.
    1. I noticed that after a day in the 4C, the solution gelled up.
    2. According to protocol, storage at temperatures below 4°C may cause precipitation of the detergents in BugBuster reagent. Incubate in a room temperature water bath with gentle swirling to redissolve.