Matt Gethers/CRI, Thailand/Labwork/Transformation

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RbCl2 Transformation

  1. Take RbCl2-competent cells (DH5 α, BL21 DE3, etc.) from the -80oC in the central bay of wing C. Each aliquot is 100 μl. Use the entire 100 μl aliquot for the reaction.
  2. Thaw cells on ice for ~10 minutes.
  3. Introduce DNA to cells and mix gently. Add 10 μl of ligation product or 1 μl of prep.
  4. Incubate on ice for 30 minutes.
  5. Heat shock at 42oC for 90 seconds on the heat block.
  6. Replace cells on ice for a few minutes.
  7. Add 1 ml SOC without antibiotic and place in the mini thermo shaker for ~1 hour at 37oC.
  8. While the cells are incubating, make up LB-Antibiotic plates with x-gal and IPTG if appropriate.
  9. Plate cells and place in incubator overnight.
  10. Colony selection the next day.

Plates

  1. Get 1.2% LB-agar from central bay area and melt.
  2. Take an aliquot for each type of plate I'll need.
  3. Add appropriate antibiotic(s). For Ampicillin, the stock solution is 1000x, so dilute x μl in x ml.
  4. After pouring the plates and allowing them to cool, spread x-gal and IPTG if doing a Blue-White Selection. The x-gal is in DMF, so it would be best to add it to LB and spread the LB. Add 20 μl x-gal and 10 μl IPTG to 70 μl LB and spread on one plate.

Run Notes

6.20.08

Ran protocol as written except 45 minute incubation on ice rather than 30 because I had to wait for the heat block to reach the correct temperature. I transformed 10 μl of ligation product into DH5 α: the ligation product of the HmgR ORF and pET-11a, the lig product between the blunt-ended pUC18 (at the SmaI locus) and the downstream fragment of HmgA (1), and the ligation product between pUC18 (at the PstI locus) and the upstream fragment of HmgR (3). Plated 100 μl of each transformation mix on amp plates (pUC18 transformations also had x-gal and IPTG), then spun down each transformation and decanted most of supernatant; resuspended and plated the pellet on another set of plates. In incubator (37) at 16:45.

Results (taken 6.21.08) - Transformants on all plates. I believe I have some white colonies on some of the pUC18 plates, but they tend to be at the edges of the plates, so I'm wondering if I just didn't get enough x-gal/IPTG at the edges. I'll wait for Mayuree to help select promising colonies.

6.27.08

Ran protocol as written . I transformed 10 μl of ligation product into DH5 α: the 6.25.08 ligation product of the HmgR ORF and pET-11a, and the 6.25.08 ligation product between pUC18 (cut at the PstI locus) and the upstream fragment of HmgR (3). I also transformed 1 μl of colony 5 prep (pET-11/HmgR ORF)into BL21 DE3. With the exception of the BL21 transformation, I plated 100 μl of each transformation mix on amp plates (pUC18 transformations also had x-gal and IPTG), then spun down each transformation and decanted most of supernatant; resuspended and plated the pellet on another set of plates. For BL21, I plated only 100 μl and saved the rest. All plates in incubator (37) at 14:45.

Results (taken 6.28.08) - Plenty of colonies on the plate with BL21 DE3 transformed with pIs001.2. Also plenty of colonies on plates (1/10 and pellet) with DH5α transformed with pIs001.3, though both appear to have less than the last transformation. Also plenty of colonies on plates with DH5α transformed with pKn002.2. Most appear blue (except for the edges), but on the 1/10 plate, there is one colony dead center (among all blue colonies) that is white. There are also 3 others near the center that are white.

7.8.08

Transformed 10 μl pKn003.L1 and pKn004.L1 into DH5α. Incubated cells on ice for 10 minutes, then added DNA, incubated for 45 minutes, then heat shock at 42 degrees for 90 seconds. Added 1 ml soc and incubated at 37 degrees for ~1 hour. Plated 100 μl and the pellet on two separate plates for each transformation (4 plates total). Plates in incubator at 1600.

Results (Taken 7.9.08) - Plenty of transformants all around. There appear to be fewer transformants than the last few attempts. For the pKn004, I suspect the lower numbers are due to less false positives (vector and insert cut with two different enzymes, so no self ligations), but I can't explain the lower numbers for the pKn003 (still a blunt-end ligation). Colony PCR results here - bad.

7.14.08

Transformed 10 μl pKn003.L2, pKn004.L2, and the pKn001 and pKn002 self ligations into DH5α (4 transformations total). Incubated cells on ice for 10 minutes, then added DNA, incubated for 45 minutes, then heat shock at 42 degrees for 90 seconds. Added 1 ml soc and incubated at 37 degrees for ~1 hour. Plated 100 μl and the pellet on two separate plates (LB Amp, 1/1000) for pKn003 and 4, but only 100 μl for the two controls (6 plates total). Plates in incubator at 1900.

Results (Taken 7.15.08) - Colonies on all plates, but control plates are comparable with experimental plates - there's a lot of background. Colony PCR results here.

7.23.08

Transformed 10 μl pKn004.L3 and pKn004.L4 into DH5α. 10 minutes on ice, added DNA, 30 minutes on ice, heat shock at 42 for 90 seconds, back on ice, added 1 ml soc, incubated at 37 for 1 hour 15 minutes. Plated 100 μl of each on LB Amp plates, then I spun down each, decanted most of supernatant (~100 μl left), resuspended the pellet, and plated. In 37 degree incubator at 1545.

Results (Taken 7.24.08) - Colonies on all plates; there appear to be less on the pKn004.L3 plate than the L4. I'm hoping that's good news. I'm doing a colony PCR and will look for clones with the right product.

7.24.08

I transformed 10 μl of pIs001.L3 into DH5α. 10 min on ice, added DNA, 30 minutes on ice, 90 seconds heat shock at 42, add 1 ml soc while on ice. Place in 37 degree shaker for an hour. Plated 100 μl and pellet on separate LB Amp plates (5 ml LA, 5 μl Amp, each plate made separately).

7.26.08

I transformed 1 μl of pIs001.P4 and P5 into BL21 DE3. 10 minutes to thaw on ice, added DNA, 30 minutes on ice, 90 seconds at 42 (used mini-incubator rather than heat blocks), added 1 ml soc and replaced on ice (a little longer than normal because the shaker had to cool), then placed in incubator at 37 degrees for one hour. Plated 100 μl of each transformation on LB Amp plates (40 ml LB-agar, 40 μl Amp, split equally between two plates). I didn't bother plating pellets as the transformations should be plenty efficient.

!It turns out I transformed into BL21, not BL21 DE3!

7.30.08

I transformed 1 μl of pIs001.P1, P3, P4 and P5 into BL21 DE3 and 10 μl of pKn005.L1 and pKn006.L1 into DH5α. 10 minutes thawing on ice, added DNA, 30 minute incubation on ice, 90 second heat shock at 42 degrees. Added 1 ml soc to each tube, then incubated at 37 degrees on shaker for 1 hour. Spread 100 μl of each on Amp/Gent plates (pKn transformations, 1/1000 each antibiotic, 80 μl each in 80 ml LB aliquotted to 4 plates) and Amp plates (pIs transformations, 1/1000 amp, 80 μl in 80 ml LB aliquotted to 4 plates). Also spun down remaining pKn transformants, decanted most of supernatant (~100 μl remaining), resuspended pellet and plated on Amp/Gen plates. Placed everything in incubator at 1700.

Results recorded 7.31.08 - For the DH5 transformations, I got one colony on the pKn006.L1 pellet plate. None of the other plates yielded any colonies. I'm doing a colony PCR on the colony. For the BL21 DE3 transformations, I got colonies on everything as usual.

7.31.08

I'm doing a fresh transformation of pIs001.P4 and P5 into BL21 DE3. 1 μl prep into cells. On ice for ~10 minutes. Heat shock at 42 degrees for ~1 min 45 seconds (a little long). Added 0.5 ml soc and immediately plated 200 μl on amp plates (master mix: 40 ml LB, 40 μl amp, aliquotted to two plates). In incubator at 1700. To be clear, there was no 1 hour incubation time after heat shock.

Results recorded 8.1.08 - Plenty of colonies on both plates. The quick and dirty worked OK.

8.1.08

I transformed 10 μl of pKn005.L2 and pKn006.L2 into DH5α. 10 minutes thawing on ice, added DNA, 30 minute incubation on ice, 90 second heat shock at 42 degrees. Added 1 ml soc to each tube, then incubated at 37 degrees on shaker for 1 hour. Spread 100 μl of each on Amp/Gent plates (1/1000 each antibiotic, 80 μl each in 80 ml LB aliquotted to 4 plates). Also spun down remaining transformants, decanted most of supernatant (~100 μl remaining), resuspended pellet and plated on Amp/Gen plates. Placed everything in incubator at 1730.

Results (Taken 8.2.08) - About 6 colonies on the pKn006.L2 pellet plate. Nothing else.

8.2.08

I transformed 10 μl of pIs001.L4 and L5 into DH5 α. I let the cells thaw for a few minutes in the ice block (not ice). I had to help along the thawing. I added the DNA and put cells on the block and the block in the fridge (I was afraid it wouldn't stay cool). When I removed the block after 15 minutes, the cells had frozen again. I helped them thaw a bit (rubbing between fingers), mixed gently with a pipette tip, and then replaced on block for a minute, then shocked at 42 degrees for 90 seconds. Replaced on ice, added 1 ml Soc to each, then incubated for 10 minutes at 37 degrees. Plated on LB Amp (master mix: 75 ml lb, 75 μl amp, aliquotted to each of four plates). Plated 100 μl, then spun down, decanted most of super, and resuspended in remaining (~100 μl) and plated. In incubator at 1900.

Results (Taken 8.3.08) - Colonies on every plate. Colony PCR Results Here.

8.5.08

I transformed 10 μl of pIs001.L6 and L7 into DH5 α. I let the cells thaw for a few minutes on ice. I added the DNA and left cells on for 15 minutes. Then shocked at 42 degrees for 90 seconds. Replaced on ice, added 1 ml Soc to each, then incubated for 10 minutes at 37 degrees. Plated on LB Amp (master mix: 80 ml lb, 80 μl amp, aliquotted to each of four plates). Plated 100 μl, then spun down, decanted most of super, and resuspended in remaining (~100 μl) and plated. In incubator at 1800.

Results (Taken 8.6.08) - Colony PCR Results Here.

8.6.08

I transformed 10 μl of pIs001.L8 into DH5 α. I let the cells thaw for a few minutes on ice. I added the DNA and left cells on for 15 minutes. Then shocked at 42 degrees for 90 seconds. Replaced on ice, added 1 ml Soc to each, then incubated for 10 minutes at 37 degrees. Plated on LB Amp (master mix: 50 ml lb, 50 μl amp, aliquotted to each of two plates). Plated 100 μl, then spun down, decanted most of super, and resuspended in remaining (~100 μl) and plated. In incubator at 1900.

Results (Taken 8.7.08) - Plenty of colonies on both 1/10 and pellet plates. Colony PCR Results Here.

8.8.08

Transformed 1 μl of pIs001.P7 and P8 into BL21 DE3. On ice block for a few minutes and helped to thaw (rubbing), added 1 μl prep, immediately shocked at 42 degrees for 90 seconds, then added 1 ml soc (while on ice block) and immediately plated 100 μl on LB amp plates (50 ml LB-Agar, 50 μl amp, split in two plates). Plates in at 2100.

8.11.08

Transformed 10 μl of pIs001.L8 into DH5α. On ice for 10 minutes to thaw, added 5 μl ligation product, let sit for 10 minutes on ice, then shocked at 42 degrees for 90 seconds, then added 1 ml soc (while on ice). Incubated for 10 minutes at 37 degrees while shaking, the plated both 100 μl and the pellet on LB amp plates (35 ml LB-Agar, 35 μl amp, split in two plates). Plates in at 1445.

Results (Taken 8.12.08) - Plenty of colonies on both plates. I selected 8 colonies to do colony PCRs. Results here.

8.12.08

Transformed 10 μl of pKn005.L3 and L4 into DH5α. On ice block for 5 minutes to thaw, added 10 μl ligation product, then immediately shocked at 42 degrees for 90 seconds, then added 1 ml soc (while on ice block). Incubated for 10 minutes at 37 degrees while shaking, the plated both 100 μl and the pellet on LB amp/gen plates (80 ml LB-Agar, 80 μl amp, 80 μl gen split in four plates). Plates in at midnight.

Results (Taken 8.13.08) - No colonies on any plates as of 0800. Will leave in the incubator today, but I don't think there's anything.

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