Transformation of plasmid into Top 10 E



Transformation of plasmid (pET100) into Top 10 E.coli cells

Day 1:

1. Get ice!

2. Remove one Top 10 cells tube (~50ul) from -80˚C. Put immediately on ice for 10 mins.

3. Allow cells to thaw on ice. Try to avoid holding the cells in your hands as much as possible as to avoid the cells from losing their efficiency.

4. Label the tube accordingly. e.g. PRL/pET100…

5. To the tube, add 1-2 ul of the ligated reaction product (PRL/pET100)

6. Mix the tube gently using pipette tip but DO NOT VORTEX!

7. Leave the tube on ice for 30 minutes. Meanwhile, thaw the SOC medium (-20˚C). Fill the heat shock wells with ¾ water.

8. Heat shock the tube @ 42˚C for exactly 40 seconds.

9. Leave the tubes on ice for 2 minutes.

10. Add 200ul of RT SOC medium to the tube.

11. Shake and incubate the tube at 37˚C for 1 hour (~250rpm).

12. Meanwhile, prepare 2 agar plates (20ul and 200ul) in the following manner:

13. To both plates, add 100ul Ampicillin (or Kianomycin depending on vector) to both plates at least 30 minutes prior to using. (If blue-white screening present, also add 40ul 100mM IPTG and 40ul X-gal). e.g. pET100 vector does not have blue-white screening, so add only Amp.

14. Once 1 hour incubation time is up, do not need to put tubes on ice anymore.

15. Prepare L-shaped Pasteur pipette for each type of plasmid.

16. To the 200ul plate, add 200ul of sample. Do not spread yet!

17. To the 20ul plate, 50ul of sample.

18. Spread the 20ul plate contents first, followed by 200ul plate (store extra sample in -4C).

19. Once, all plates have been prepared, leave in incubator at 37˚C (Dr. Stiller’s lab) for 20-30 minutes.

20. After 20-30 minutes, turn plates upside-down and leave overnight!

Day 2:

1. Check for blue and white colonies.

2. Select 5-10 white colonies. To be performed under flame to avoid contamination.

3. Inoculate white colonies using toothpick into 10mL LB with 10ul Ampicillin.

4. Shake at 37˚C overnight (~10hrs) at 225rpm (N-401).

5. NOTE: For Top 10 cells, don’t follow steps 5 & 6.

6. When expressing proteins (specifically use BL21 E.coli cells), check O.D.600 until it reads between 0.4-0.8, add 100ul 100mM IPTG (inducer).

7. Take 500ul samples at 0h, 4h, 12h, 24h…..(spin down and discard supernatant. Store in -20˚C) to check on SDS-PAGE gel.

Day 3:

1. Make Glycerol stocks (300ul 50% autoclaved glycerol + 700ul cells after vortexing) of all samples. Store in -80˚C.

2. Perform Miniprep.

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