Electrotransformation of BMH 81-17mut S for Isolating Site-Directed dsDNA Mutants

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Electrotransformation of BMH 81-17mut S for Isolating Site-Directed dsDNA Mutants

Date Added: May 27, 2008 05:54:46 AM

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Category: Bacteria Transformation Protocols

Picking Transfected ES Cell Clones Protocol

Picking Transfected ES Cell Clones - Day 14

1. Thaw the electrocompetent cells on ice.

2. Chill the cuvettes and the microcentrifuge tubes containing the DNA.

3. In a 15 ml sterile centrifuge tube, mix 1 ml of LB Electroporation Solution.

4. Check the set-up of electroporation equipment. Make sure the pulse controller is plugged in and set at 400 . Set the capacitance at 25 F. Set the voltage at 2.5 kV (maximum).

5. Just before the electroporation of each sample, fill a Pasteur pipet and bulb with the 1 ml of LB mix in the 15 ml tube.

6. Take the cells up in a 200 l tip, mix with the DNA and transfer to the cuvette by emptying the tip down one side of the cuvette. (Hint #1).

7. Wipe off any moisture on the sides of the cuvette.

8. Place the cuvette in the cuvette holder and press both red buttons simultaneously until the beeper sounds. (Hint #2).

9. Let the cells shake at 255 rpm for 45 to 60 minutes at 37°C.

10. After use, immediately rinse the cuvettes thoroughly with ddH2O and 95% Ethanol. Let the cuvettes air dry.

11. Plate 10 l of the cell suspension onto the LB plates containing the appropriate antibiotic for the plasmid. (Hint #3).

12. Add the rest of the 1 ml suspension to 5 ml of LB containing the appropriate antibiotic. Add 2.5 ml to one sterile tube, and the rest to another sterile tube.

13. Grow the cultures overnight (12 to 16 hours) at 37°C in a shaking incubator at 225 rpm.

14. Aliquot 1 ml per microcentrifuge tube for a total of 4 microcentrifuge tubes (see Hint # 4).

15. Prepare the plasmid DNA by the miniprep procedure. (See protocol on Plasmid DNA Preparation). Resuspend the plasmid DNA in a volume of 75 l of TE.

16. Digest 1 l of the miniprep using 40 units of the restriction enzyme that represents the unique site in the original plasmid in a total volume of 40 l. (see Protocol on Restriction Enzyme Digestion).

17. Incubate the digestion reaction for 2.5 to 4 hours.

18. Electrophorese 2 l of the restriction digest mix on an agarose gel to check that the sample appears fully digested. (see Protocol for Electrophoresing DNA on an Agarose Gel).

19. Purify the DNA in the rest of the reaction mix (see Protocol on Purification of Plasmid DNA) (see Hint #5).

20. Use 0.3 l for transformation into DH5 cells (See Protocol on Transformation). Plate 5 l and 50 l onto LB plates containing the appropriate antibiotic.

21. Pick colonies and isolate the plasmid DNA. (See Protocol on Plasmid DNA Isolation).

22. Digest the DNA with a restriction enzyme that will not cut the newly mutated plasmid. (See Protocol on Restriction Enzyme Digestion).

23. Sequence those clones that are not digested by the specific restriction enzyme whose site has been mutated (see Protocol on Sequencing DNA). These are the clones that should contain the site-directed mutation (Hint #6).

Solutions and Buffers

BMH 81-17 mut S cells BMH 81-17 mut S cells BMH 81-17 mut S electrocompetent cells

See Protocol on making electrocompetent cells

TE Buffer 10 mM Tris

pH 8.0

1 mM EDTA

LB Plates

LB Plates Store at 4°C until needed
15 g/liter Bacto-agar
Let cool to 50°C before adding any antibiotics.
Prepare in LB
Swirl flask and pour approximately 30 ml/petri plate (100 mm).
Let the media set.
95% (v/v) Ethanol
Sterilize by autoclaving for 20 minutes at a pressure of 15 pounds/square inch on the liquid cycle.

Luria Broth (LB) 5 g/liter NaCl
5 g/liter Yeast Extract
10 g/liter Tryptone
1 ml/liter 1 M NaOH

1 M NaOH

LB Electroporation Solution 1 ml of LB Buffer
10 mM Magnesium Sulfate
0.4% (w/v) Glucose

DH5α Cells

DH5α Cells DH5α electrocompetent cells
See Protocol on making electrocompetent cells

BioReagents and Chemicals:

EDTA
Magnesium Sulfate
Tris
Sodium Hydroxide
Sodium Chloride
Bacto-Agar
Yeast Extract
Tryptone
Antibiotic
Ethanol
Glucose

Protocol Tips:

1. Tap the cells down to the bottom of the cuvette so that the cells form an even layer from one side to the other. Try to avoid any large air bubbles.

2. The time constant should be 7.0 to 7.5 msec as less than 6.5 msec gives reduced viability, less than 4 gives a small explosion, generally from too high an ionic strength in the samples. If the time constant is greater than 8, the efficiency of the electroporation is low. If any of these conditions result, take a fresh cuvette, sample, cells and try again. Otherwise, check the competency of the cells and repurify the DNA.

3. This step is used to check the electroporation efficiency. The electroporation frequency of linear DNA is about a thousand-fold less efficient than circular plasmid DNA. Since a considerable portion of the DNA will be linear using this protocol, the overall efficiency per microgram of DNA will be less than the usual expected efficiency.

4. If you have many different mutants, two microcentrifuge tubes per mutant clone will suffice.

5. The contributors of this protocol purify the DNA by loading the rest of the restriction digest mix onto a Biogel P10™ column. Purify according to manufacturer's recommendations.

6. For single base changes in the target DNA, usually 100% of the selected minipreps will have the right mutated sequence. For more complicated mutations, this frequency varies from 10 to 100%.

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