Re-transform with selected plasmid

From 2008.igem.org

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(New page: ==Transformation Protocol== Before you start, make sure the water bath is at 42 degrees and the SOC is at 37 degrees. If the SOC is cloudy or has anything floating in it, it has gone bad ...)
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==Transformation Protocol==
==Transformation Protocol==
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Before you start, make sure the water bath is at 42 degrees and the SOC is at 37 degrees. If the SOC is cloudy or has anything floating in it, it has gone bad and you'll have to make more. Also, make sure you have enough plates and check the resistance of the DNA before plating.  
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Transformations are used to insert the designed DNA into bacteria, which is then grown to multiply the plasmid DNA.
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1. All bacterial cells are found in the -80 in the top section. For ligations, use ultracompotent cells. For all other transformations use excel10 cells. Thaw the cells and the DNA on ice for 7-8 min.
 
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2. Take 25 ul of the cells and place them in a different 2ml microcentrifuge tube. Add 1ul of DNA for regular transformations or 3ul-5ul of DNA for ligation transformations. Let the tubes sit on ice for 30 min.
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'''Heat Transformation Protocol'''
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3. Place the tubes in the water bath for exactly 30 sec, and then place them on ice for 2min.
 
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4. Put 900ul SOC in each tube and place in the 37 degree shaker for 60 min.
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1. Make sure that the incubator (30/37C) and water bath (42C) are ON
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5. Take tubes out of the shaker and centrifuge at 6,000rpm for 1min. You should see a white pellet at the bottom.  
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2. Make sure required antibiotic plates are present. Check the antibiotic resistance on the plasmid map in Vector NTI.
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6. Remove .85ml from the each tube and discard. Resuspend the remaining 50ul.
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3. Take the DNA out of –20 frig, let it thaw
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7. Plate the 50ul. First, place the transformed cells on the plates. Then spread the cells across the entire plate.  
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4. Thaw the competent cells on ice for 7-8 min.
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8. Place the plates upright in the incubator for 20 min. After this, flip the plates and leave them in the incubator for 12-14 hrs.
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5. Add 1.0 µl of DNA (about 10ng) into the liquid (Don’t vortex). Tap the sides of the tube to mix. For Ligation add 1-5 ul
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6. Incubate the cells on ice for 30 min
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7. Heat shock the cells for EXACTLY 30 sec at 42 C water bath.
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8. Place on ice for 2 min.
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9. Add 0.9ml of 37˚ C S.O.C medium to each tube (S.O.C is made by dissolving 0.5 ml of 20% glucose in 25 ml of SOB. Make sure that the SOC is clear and not cloudy/ contaminated.)
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10. Shake the tubes at 37 C, 280 rpm for 60 min or 30 C for 90 min
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11. Spin down the cells at 6000 rpm for 1 minute (should see white clumps at bottom).
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12. Take out 0.85 ml of S.O.C. (so there’s only 50 ul of SOC left inside)
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13. Resuspend the cells.
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14. Plate all 50 ul.
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15. Incubate upright for 20 min., then upside down overnight (12-14 h) at 37 C or 16-18h at 30C.
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Can leave the cells in the incubator for up to 18 hours but no more

Revision as of 10:39, 29 October 2008

Transformation Protocol

Transformations are used to insert the designed DNA into bacteria, which is then grown to multiply the plasmid DNA.


Heat Transformation Protocol


1. Make sure that the incubator (30/37C) and water bath (42C) are ON

2. Make sure required antibiotic plates are present. Check the antibiotic resistance on the plasmid map in Vector NTI.

3. Take the DNA out of –20 frig, let it thaw

4. Thaw the competent cells on ice for 7-8 min.

5. Add 1.0 µl of DNA (about 10ng) into the liquid (Don’t vortex). Tap the sides of the tube to mix. For Ligation add 1-5 ul

6. Incubate the cells on ice for 30 min

7. Heat shock the cells for EXACTLY 30 sec at 42 C water bath.

8. Place on ice for 2 min.

9. Add 0.9ml of 37˚ C S.O.C medium to each tube (S.O.C is made by dissolving 0.5 ml of 20% glucose in 25 ml of SOB. Make sure that the SOC is clear and not cloudy/ contaminated.)

10. Shake the tubes at 37 C, 280 rpm for 60 min or 30 C for 90 min

11. Spin down the cells at 6000 rpm for 1 minute (should see white clumps at bottom).

12. Take out 0.85 ml of S.O.C. (so there’s only 50 ul of SOC left inside)

13. Resuspend the cells.

14. Plate all 50 ul.

15. Incubate upright for 20 min., then upside down overnight (12-14 h) at 37 C or 16-18h at 30C. 


Can leave the cells in the incubator for up to 18 hours but no more
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