Matt Gethers/CRI, Thailand/Labwork/HmgR Isolation Protocol

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HmgR Isolation Protocol

Mini Isolation Protocol

  1. Transform 1 μl of pIs001 into BL21 DE3 for expression. These transformants aren't very stable, so I think I'll have to transform anew every time I want to isolate the HmgR protein.
  2. Inoculate a 5 ml culture in LB Amp (10 μl of stock brings to 200 μg/ml) and 100 μl 20% glucose (brings to 0.4%) to repress expression. Be sure to take two 1 ml samples for blanks before adding the cells. Grow up the culture to between OD 0.4 and 0.8, then split into two 2.5 ml cultures.
  3. Add 12.5 μl IPTG to one of the two 2.5 ml cultures (brings to 1 mM). Grow for 2 hours (cells should be approximately the same density after so short a time).
  4. Spin down the cells at 10,000 RPM for 5 minutes, decant supernatant, and resuspend in 200 μl 50 mM phosphate buffer.
  5. Sonicate the cells to rupture.
  6. Spin down the lysed cells at 10,000 RPM for 5 minutes.
  7. Pipette off the supernatant and put in an Eppendorf.
  8. Wash the pellet in 200 μl 50 mM phosphate buffer, spin down again at 10,000 RPM for 5 minutes, and resuspend in 200μl PB one more time.
  9. I should do a Bradford assay on both fractions to determine approximately how concentrated my samples are so I can load approximately the same amount of protein in each lane of the SDS-PAGE.

Run Notes

7.2.08

Yesterday evening at 1845, I started a 2 ml culture of pIs001.2 in BL21 in LB/Amp/Glucose (2 ml LB/4 μl Amp/40 μl 20% Glucose). This morning, I made up 9 ml tubes of the same media (10 ml LB/20 μl Amp/200 μl 20% Glucose) and took 1 ml from each for blanking. Although they are made of the same media, the spec requires two 1 ml blanks, so that's why I took two mls. To each 9 ml tube, I added 90 μl overnight culture (1/100) and put in the 37 degree shaker at 1030. I'll check the OD at 1115. ODs for both cultures at 1115 were ~0.09. I'll check again around 1150 (before lunch).

7.2.08 OD Log

7.29.08

This morning, I started 4 4 ml cultures of pIs001.P5 and P6 in BL21 (2 cultures each). I made a master mix of 20 ml LB, 40 μl Amp, and 400 μl 20% glucose, took 2 1 ml aliquots for blanks, and aliquotted 4 ml to each of 4 tubes. I then selected 4 colonies (again, 2 for each clone) and inoculated in 50 μl water. I used 25 μl of this suspension to inoculate the 4 ml tubes. In shaker at 0920. These cultures will probably grow more slowly than the the 7.2.08 cultures as I'm inoculating directly from the plate.

7.29.08 OD Log

The cultures had overgrown by 1300 (see log), so I diluted each culture 1 in 3 into fresh media (4 ml LB, 8 μl Amp, 80 μl 20% Glucose per tube, made master mix of 16 ml LB, 32 μl Amp, 320 μl 20% glucose and aliquot into four tubes). 2 ml culture in 4 ml fresh media and then replaced for 25 minutes. Removed at 1345 - most cultures in the correct range (see log). I split only P5 #1 and P6 #1 culture in two (approximately 3 ml each) and added 15 μl 200 mM IPTG to one half (2 of 4 tubes). Incubated for 2 hours in the shaker, then transfered to centrifuge tubes, spun down at 6000 RPM at 4 degrees C, decanted super, and froze in the -20 over night.

Morning of 7.30.08, I resuspended each pellet in 300 μl 50 mM PB, no PMSF, no DTT. I sonicated, spun down for 5 minutes at 10,000 RPM, collected supernatants. Then I washed the pellets again in 300 μl PB and finally resuspended in the same. I took 15 μl of each sample (8 total supernatant/pelletm -/+ IPTG, P5/P6) and added 3 μl loading buffer and boiled at 95 degrees C for 5 minutes. Loaded on gel. Gel results here.

7.31.08

This morning I started 3 2 ml cultures of pIs001.P1, P5, and P6. I made a master of 6 ml, 12 μl Amp, 120 μl 20% glucose, then split into 3. Inoculated each with an entire colony via loop.

7.31.08 OD Log

At 1020 I split each culture into two 1 ml cultures and added 5 μl IPTG to 3 of 6 tubes. Grew for 2 hours (in at 1045, out at 1245). At 1250, I removed cultures, spun down at 10,000 RPM for 5 minutes (not at 4 degrees), and placed on ice. I decanted the super and resuspended each pellet in 300 μl PB. Sonicated. Spun down at 10,000 RPM for 5 minutes again (not at 4 degrees), and moved supernatant to a separate tube. I washed the pellet in 300 μl PB. I then took 15 μl of -/+ IPTG Pellet/Super P5, -/+ IPTG Super P6/P1 (8 samples total) and added 3 μl loading buffer. Boiled for 5 minutes and spun down, then loaded on a gel. Gel results here.

8.1.08

This morning I started 3 2 ml cultures of pIs001.P1, P5, and P6. I made a master of 6 ml, 12 μl Amp, 120 μl 20% glucose, then split into 3. I inoculated P1 with 1 colony, but inoculated P5 and P6 with many colonies.

8.1.08 OD Log

At 1115 I removed the cultures and checked the ODs - about right. I split each culture in 2 (1 ml each) and added 5 μl 200 mM IPTG to the "+ IPTG" tubes. Placed in shaker at 1125 and will removed at 1325. Spun down the cells at 10,000 RPM for 5 minutes, decanted super, resuspended each of the 6 pellets in 300 μl 50 mM PB. Placed on ice and sonicated. Spun down sonicated cells at 10,000 RPM for 5 minutes, took supernatant and placed in a separate tube. Washed pellets in 300 μl PB. Added 15 μl of sample to 3 μl of loading buffer and boiled for 5 minutes at 95 degrees. Loaded 5 μl on a gel of the following: P5 -/+ Pellet/Supernatant, P6 -/+ Supernatant, P1 -/+ Supernatant (8 total). Gel results here.

8.9.08

This morning I started two 2 ml cultures of pIs001.P7 and P8. I made a master of 4 ml, 8 μl Amp, 80 μl 20% glucose, then split into 2. I inoculated each with one colony from the 8.8.08 transformation into DE3.

8.9.08 OD Log

At 1200, I split each culture into two 1 ml cultures, and added 5 μl 200 mM IPTG to the "+ IPTG" samples. Collected at 1400. Spun down at 10,000 for 2 minutes at room temp, decanted supernatant, and resuspended each pellet in 300 μl 50 mM PB. Took 15 μl of each and added 3 μl 6x loading buffer. Boiled for 5 minutes at 95 degrees C, then loaded on a poly-ac gel. Gel results here.

En Masse Isolation Protocol

  1. Transform 1 μl of pIs001 into BL21 DE3 for expression. These transformants aren't very stable, so I think I'll have to transform anew every time I want to isolate the HmgR protein.
  2. Inoculate a 200 ml culture in LB Amp (400 μl of Amp stock brings to 200 μg/ml) and 4 ml 20% glucose (brings to 0.4%) to repress expression. Grow up the culture to between OD 0.4 and 0.8, then take a 1 ml sample for a dignostic gel.
  3. Add 24 mg IPTG to the flask (brings to 0.5 mM). Grow for 2 hours. Take a 1 ml sample for a diagnostic gel.
  4. Pour cultures in centrifuge tubes and spin down to collect the cells 2 pellets - then combine.
  5. Resuspend each pellet in 1 ml low salt buffer supplemented with the protease inhibitor cocktail and DTT (10 ml low salt buffer, 10 μl 1 M DTT, 1 pellet cocktail).
  6. Run the cells through a French Press and collect the lysate or sonicate and spin down.
  7. Pool the supernatants and resuspend the pellets and pool in a separate tube.
  8. The supernatant sample will be used in the purification protocol.

Run Notes

7.4.08

Yesterday evening at 1700 I started a 5 ml culture (5 ml LB, 10 μl Amp, 100 μl 20% glucose). This morning, however, I noticed that the culture was contaminated, so I inoculated a 200 ml culture (LB, Amp, Glucose as written in protocol above) directly from the plate of BL21 transformants (6.27.08 transformation) at 0930. The culture will probably take quite some time to grow up, so I only expect to get to spinning down the culture into pellets after induction by the end of the day.

7.4.08 OD Log

I removed the culture at 1530 and took a 1 ml sample for later analysis. I then added 240 mg IPTG to the 200 ml culture, then replaced in shaker at 1600.

7.27.08

Yesterday evening I transformed pIs001.P5 and pIs001.P6 into BL21. This morning, I collected the colonies and used half a colony from the P5 plate to inoculate a 200 ml culture (200 ml lb, 400 μl amp, 4 ml 20% glucose). In shaker at 0940. I expect this will grow up a bit faster than last time as the cells are fresh from the 37 degree incubator, so I'll plan to check again at 1 or so.

I just realized that 240 mg IPTG in 200 ml culture is 5 mM, not 0.5 mM. However, what I did last time seemed to work, so I'm going to do the same thing.

7.27.08 OD Log

I removed the cells at 1640, took a 1 ml sample to run on the gel later, and added 240 mg IPTG. In at 1545. Will remove at 1745 to spin down.

Removed cells at 1745, took a 1 ml sample for the gel, then aliquotted the remaining culture to 6 centrifuge tubes. Spun down at 6000 RPM for 5 minutes at 4 degrees. I spun the 1 ml samples at 1000 rpm for 3 minutes at room temp. Note: the pre-induction sample was on my benchtop all this time. After all spinning was done, I decanted the supernatant and froze in the -20.

To determine

  • Stock concentration of glucose; where is glucose?
    • 20% glucose in fridge on door shelf.
  • Molar concentration of IPTG?
    • 200 mM.
  • Where is the phosphate buffer and what is it's concentration?
    • On my bench (literally right in front of me) at 50 mM.
  • Sonication how to.
    • PiKoy will teach me when I'm ready to use it.
  • How long to spin down cells after lysing?
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