1996

Also, see this protocol and Measuring transformation efficiency.

30 mm potassium acetate 1.47 g

50 mm manganese chloride 4.95 g

100 mm rubidium chloride 6.05 g

10 mm calcium chloride 0.56 g

15% (w/v) glycerol (15 x 5) 75 ml

1. Bring the volume up to 500 ml using ddw.

2. Adjust the ph with 0.2 n acetic acid to ph 5.8. Throw the solution out if the ph is less than 5.8. (i.e. 5.82 or so is useful)

3. Sterile filter the solution and keep at 4 deg.

10 mm mops ph 7.0 0.42 g

75 mm calcium chloride 1.66 g

10 mm rubidium chloride 0.24 g

15% (w/v) glycerol 30 ml

1. Bring the volume up to 200 ml by adding ddw.

2. Adjust the ph with 0.2 n acetic acid to ph 5.8. Throw the solution out if the ph is less than 5.8. (i.e. 5.82 or so is useful)

3. Sterile filter the solution and keep at 4 deg.

Tb (transformation buffer) - found in the molecular biology refrigerator #1

Polystyrene tubes (cat # 2057)

Sterile eppendorf tubes (microliter tubes)

37 or 42 deg water bath

Falcon tube (cat # 2070)

Use only disposable pipets

1. Always wash your hands and spray with 70% etoh or wear gloves.

2. Always keep the caps on the polystyrene tubes loose so that air can circulate. Only cap tightly when the cells are no longer growing and are being stored in the refrigerator until the transformation efficiency has been calculated.

3. When scraping the frozen cell sample, hold the eppendorf tube at the top rather than at the bottom, so that the sample does not fully thaw. (one may want to keep the frozen samples on dry ice.)

4. When doing this experiment always have a negative control. A negative control is used to show that the media is not contaminated and what is growing in the media are the bacteria cells and not contaminants.

5. Always make sure the shaker is turned on when you leave the bacteria warmroom.

1. Decide which competent cells to make.

2. Make the appropriate # of agar plates plus one extra small agar plate (100 mm) to act as a control for the media ( shows that the media is not contaminated) - refer to method for making gel plates. Do not use antibiotics in these agar plates.

3. Using the cell record notebook labeled bacterial stocks i: libraries, vectors, and hosts, locate the cells which are of interest in the -80 deg freezer in room #524 (if you have never done this please ask Subha for help the first few times).

4. After you have located the cells in the notebook, get a ice bucket and fill it with ice. Using the cell records, obtain the appropriate vials from the freezer. Be sure to keep the cells on ice at all times and when holding the vials, to hold the vials as far away from the cell sample as possible (this will prevent the cells from melting too quickly by being warmed by one�s fingers).

5. Spread one type of cell/plate. Be sure to mark the bottom of the agar plate appropriately.

6. To spread the cells: using a sterile tooth pick or a sterile innoculating loop, lightly touch the top of the cell sample in the vial (the cells should not be completely thawed nor should such a large amount of the sample be taken that it can be clearly seen on the loop or the tooth pick ). Quickly and lightly spread the cells on the agar plate.

7. Allow the cells to grow in the bacteria warm room overnight, room #539. The plate should be upside down in the warm room so the condensed water droplets do not fall on the gel.

1. Grow a single colony in a polystyrene tube (cat #2057) picked from an agar plate at 37 degc in 3 ml lb broth.

2. Pick the colony using a sterile tooth pick or a sterile pipet tip.

a. Mark the colony which one is going to pick; the colony should no be attached to other colonies.

b. Pick the colony by gently touching the tooth pick or the pipet tip to the desired colony.

c. Drop the tooth pick or the pipet tip into the polystyrene tube with the lb broth. Leave the tip in the tube over night.

d. Be sure to label the tube with the strain, the date and the lab room #, and your name.)

3. Allow the culture to grow overnight on the shaker @ 265-275 rpm.

1. Take 2 ml of of the above 3 ml cell culture and dilute it into 50 ml of SOB contained in a 250 ml flask (cells need a lot of air to grow and multiply). Grow the cells on the shaker in the warm room (250 to 270 rpm) until od 600 = 0.45 (1 to 2 hours)

2. Check the cells for the first time after one hour has passed and from then on every 1/2 hour until the count is close to 0.45 - then count the cells every 15 min.

3. Chill the cells on ice for at least 10 to 15 min. (max. 1 hour ) to prevent further growth.

4. Spin the cells down in a 50 ml falcon tube (cat # 2070) at 2.5k rpm for 15 min. At 4 deg.

5. Remove the supernatant using a sterile pipet.

6. Resuspend each tube with 16 ml of ice cold tb by gentle and brief vortexing. Use a new pipet for each addition; one doesn�t want to cross contaminate the different cell lines. Vortex @ 5 to get the pellet off the bottom. Reduce the speed to 3 or 4 to mix the pellet with the tb.

7. Allow the cells to sit in the ice for 10 min. Before spinning cells down again.

8. Spin at 2.5k rpm for 15 min. At 4 deg.

9. Remove supernatant using a sterile pipet.

10. Resuspend each tube gently (by pipeting) with 4 ml of ice cold tb. Gently vortex with finger (tap finger against the bottom of the test tube).

11. Add DMSO to 7% concentration (add drop-wise while swirling). add 280 ul of DMSO to 4 ml of tb to produce a 7% concentration of DMSO (dimethyl sulfoxide, light sensitive).

12. Aliquot 0.2 ml (200 ul) of the solution using a sterile pipet into 1.5 ml eppendorf tubes which have been chilled on ice and then freeze the cells at -80 deg. Be sure to make adjustments in competent cell notebook.

Be sure to have large or small (150 mm, cat # 1058; 100 mm, cat # 1029) lb agar plates which have been spread with both x-gal & iptg if blue/white color selection is appropriate for the vector being added and the appropriate antibiotic has also been added to the lb media with agar. They must be prepared one hour before use. Spread 45 ul, 40 mg/ml x-gal, and 45 ul, 0.5m IPTG if using a large plate or spread 30 ul, 40 mg/ml x-gal, and 30 ul, 0.5m iptg if using a small plate . Both x-gal and IPTG can be found in the - 20 deg freezer, molecular biology #1.

Note that both x-gal and IPTG are light sensitive and therefore should only be taken out of the light proof box for as brief a time as possible.

1. If cells are frozen thaw aliquots on ice until liquid and then keep on ice for 5 to 10 min (up to 1 hr).

2. Add DNA in less than 50 ul per 200 ul of cells (use less than 0.1 ug per 200 ul of cells). for example: if pgem 7zf (+) is @ a concentration of 1 ng/ul , add 20 ul of dna per 200 ul b/c you would be adding 20 ng / 20 ul which is the same as 0.02 ug per 200 ul.

3. Leave on ice for 20 min.

4. Heat shock the cells for 30 sec. At 37 deg c (the Okayama paper showed that the temperature is not critical for heat shock [one can go as high a 52 deg and as low as 30 deg] but that the time does make a difference, 30 sec being the ideal; refer to page p.26 fig.4; a copy of the article is in the last section of the competent cell notebook.)

5. Keep the cells on ice for 2 min.

6. Mix 0.8 ml (800 ul) of SOC media and the 0.2 ml of culture in a polypropylene tube. Place the tube in the slow shaker (on right side as you walk in the room) in the bacteria warm room (210 to 220 rpm) for 1 hr.

7. Streak 0.2 ml of culture onto each large lb agar plate which has been coated with x-gal & IPTG if appropriate and an antibiotic. Use a bent pasteur pipet to smooth the cells evenly over the plate. Be sure to shake the cells gently before plating to prevent settling. The plating of the cells must be done quickly so they are evenly absorbed throughout the plate.

8. Allow plate(s) to sit for 10 to 30 min. Look to see if all of the culture has been absorbed.

9. Place plates in warm room overnight upside down so the condensed water does not collect on the culture plate.

10. Count cells. If the cells are spread relatively evenly over the plate, one can count the cells by dividing the plate into quarters, counting one quarter of the plate and then multiplying by four.

11. Calculate the efficiency (no. of colonies / ng DNA)

Strain

Date prepared

Checked with vector

Efficiency

Box number

Slot number

Efficiency after freezing

Experiment number

Cells

Box number

Slot number

Absorbance at 600 nm

Antibiotic

Concentration of antibiotic added to gel

Blue/white color selection

Amount of dna added

Transformation efficiency

Observations