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From 2008.igem.org

Contents 1 Plasmid DNA purification 1.1 Materials 1.2 Procedure 2 Restriction Digest 2.1 Materials 2.2 Procedure 3 Agarose Gel electrophoresis 3.1 Materials 3.2 Procedure 4 Making electrocompetent cells for immediate use, Procol Aus Berlin 4.1 Materials 4.2 Procedure 5 Making electrocompetent cells for stock 5.1 Materials 5.2 Procedure 6 Making your own Glycerol Stock 6.1 Materials 6.2 Procedure 7 Electro-transformation 7.1 Materials 7.2 Procedure 8 Taq PCR 8.1 Mix 8.2 Cycles 9 Pfx PCR 9.1 Mix 9.2 Cycles 10 Ligation 10.1 Materials 10.2 Procedure 11 PCR with end filling 11.1 Materials 11.2 Procedure

Plasmid DNA purification

Materials

• LB broth with appropriate antibiotic
• 15 ml tube
• incubation oven at 37°C
• microcentifuge tubes
• table-top microcentrifuge
• buffer P1 (Qiagen kit)
• buffer P2 (Qiagen kit)
• buffer N3 (Qiagen kit)
• QIAprep spin columns
• transparant tape
• buffer PE (Qiagen kit)
• buffer EB (Qiagen kit)
• nanodrop

Procedure

1. Inoculate a single colony into 5 ml of LB with the appropriate antibiotic and incubate at 37°C for 12-16 hours (liquid culture).
2. Put 1.5 ml of this liquid culture in a microcentrifuge tube and centrifuge at 8500 rpm for 3 minutes at room temperature.
3. Remove all traces of supernatant by inverting the tube.
4. Resuspend the pelleted cells in 250 µl Buffer P1 and vortex until no cell clumps remain.
5. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times. !! Do not vortex and do not allow the reaction to proceed for more than 5 minutes !
6. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. The solution will become cloudy.
7. Centrifuge for 10 minutes at 13000 rpm. A compact white pellet will form.
8. Apply the supernatant of step 4 to a QIAprep spin column by decanting. When you label the columns, cover this label with tape (the ink tends to dissolve).
9. Centrifuge for 1 minute at 13000 rpm. Discard the flow-through. If you made several tubes of one sample, you can repeat the steps 8 and 9 in the same QIAprep spin column. This way, the concentration of plasmid DNA will be higher.
10. Wash the QIAprep spin column with 0.75 ml Buffer PE. Centrifuge for 1 minute at 13000 rpm. Discard the flow-through !!
11. Centrifuge for an additional minute to remove all residual wash buffer.
12. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 µl Buffer EB to the centre of the QIAprep spin column, let stand for 1 minute and centrifuge for 1 minute at 13000 rpm.
13. Measure the concentration of plasmid DNA with the nanodrop (ng/µl). Use Buffer EB as blank.

Restriction Digest

Materials

• restriction enzymes ( EcoRI, SpeI, PstI and XbaI)
• restriction buffer H
• mQ
• plasmid DNA
• blue juice
• Smart ladder (reference)

Procedure

Here we describe a 20 µl reaction. The used restriction enzymes are from Roche. Prepare several tubes of the same sample.

• If you want to digest with a mixture of EcoRI and SpeI, add the following to a microcentrifuge tube:
  • 500 ng plasmid DNA
  • (14-X) µl mQ
  • 2 µl buffer H: Vortex buffer before pipetting to ensure that it is well-mixed.
  • 1 µl EcoRI and 1 ul SpeI. Vortex enzyme before pipetting to ensure that it is well-mixed. Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 ul, just touch your tip to the surface of the liquid when pipetting. The restriction enzymes must be the last thing you add. Allways keep them on ice.
• If you want to digest with a mixture of EcoRI and XbaI, add the following to a microcentrifuge tube:
  • 500 ng plasmid DNA
  • (17-X) µl mQ
  • 2 µl buffer H: Vortex buffer before pipetting to ensure that it is well-mixed.
  • 1 µl XbaI: Vortex enzyme before pipetting to ensure that it is well-mixed. Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 ul, just touch your tip to the surface of the liquid when pipetting. The restriction enzymes must be the last things you add. Always keep them on ice.

1. Incubate the tubes at 37°C for 1.5-2 hours (heat block or oven). In the mean time, you can prepare the agarose gel.
2. For the digest with XbaI: put 2 µl of DNA with 8 µl mQ and 2 µl blue juice on gel to check whether the enzym has properly cut. Then, add 1µl EcoRI to the rest of the mixture and incubate for 1.5-2 hours at 37°C.
3. Add 4 µl blue juice to each tube.
4. Load 5 µl reference mixture (ladder) onto the gel.
5. Load 25 µl digest onto the gel. Make sure that you have multiple lanes with the same BioBrick (higher concentration).
6. Start the electrophoresis (this should take about 1 hour). Look at the result under UV-light and cut out the correct fragment.
7. Purify the obtained fragment.

Agarose Gel electrophoresis

Materials

• Agarose
• 200 ml 1x TBE Buffer
• erlenmeyer flask (500 ml)
• microwave
• microcentrifuge tubes
• electrophoresis apparatus
• ethidium bromide
• gloves

Procedure

1. Dissolve 2 g Agarose into 200 ml 1x TBE Buffer. This way you will obtain a 1% agarose gel.
2. Heat this mixture in the microwave oven for 3-4 minutes (position of the button is "cuisson"). Stir or swirl from time to time.
3. Clamp the gel rack in the holder and add 2 drops of Ethidium Bromide. Also insert the comb. Use gloves!
4. When the melted Agarose has cooled down, you can pour it into the gel rack. Mix the EtBr with the gel using the comb. No gloves!
5. Wait until the Agarose is properly jellified (15 minutes).
6. Put the gel rack with the gel inside the electrophoresis tank. Fill the tank with 1x TBE Buffer and remove the comb. (DNA moves towards the positive anode, which is the red side). Use gloves!
7. Now you can load the samples (25 µl). No gloves!
8. Put the lid onto the apparatus (gloves!) and start the electrophoresis (no gloves): set > set > 125V > 500mA > 1h > run. You should see some bubbels near the electrodes.
9. After 1 hour, stop the electrophoresis, remove the lid and take the gel rack to the UV lamp.
10. Take a look at the gel under UV radiation. Wear eye protection!
11. You can cut out the part of the gel that you need for later experiments.

Making electrocompetent cells for immediate use, Procol Aus Berlin

Materials

• ice
• LB broth with the appropriate antibiotic
• 15 ml tube
• OD meter
• cooled microcentrifuge (4°C)
• mQ
• cuvette
• Gene Pulse apparatus

Procedure

1. Grow cells to OD 0,5 (no preculture needed, just inoculate from plate in the morning).
2. Put cells 10 min on ice.
3. Centrifugate 2ml cells in 2 ml eppendorf tubes, 2 min on 11000 rpm (attention: centrifugate below 4°C!).
4. Wash two times with 2 ml mQ (0°C).
5. Wash a third time with 1 ml mQ (0°C).
6. Resuspend pellet in 40 ul mQ
7. Electroporate in 1 mm cuvette at 1,8 kV.

Making electrocompetent cells for stock

Materials

• 500 ml LB broth
• overnight culture of cells
• ice
• cooled centrifuge
• 750 ml autoclaved water
• 10% glycerol

Procedure

1. Inoculate 500 ml of LB broth with 5 ml of a fresh overnight culture.
2. Grow cells at 37°C with vigorous shaking to an ABS600 of 0,5 to 1,0 (the best results are obtained with cells that are growing rapidly; the appropriate cell density, therefore, depends on the strain and growth conditions).
3. To harvest, chill the flask on ice for 15 to 30 minutes, an centrifuge (2 x 250ml) in a cold rotor at 4000g for 15 minutes.
4. Remove as much of the supernatant (medium) as possible. Resuspend pellets in a total of 2 x 250 ml of cold water. Centrifuge as described in previous step.
5. Resuspend in 125 ml of cold water (pour two centrifuge tubes together to obtain 250ml). Centrifuge as described in previous step.
6. Resuspend in 10 ml of cold 10% glycerol. The cell concentration should be about 1 - 3 x 10^10 cells/ml.
7. This suspension may be frozen in aliquots on dry ice, and sotred at -70°C. The cells are good for at least 6 months under these conditions.

Making your own Glycerol Stock

Materials

• LB broth with appropriate antibiotic
• 50% glycerol
• cryotubes

Procedure

1. Pick a single colony from the above plate into 5 ml of LB broth with appropriate antibiotic and grow 12-14 hours to create an overnight liquid culture.
2. Combine 900 µl of overnight culture and 900 µl of a sterile 50% glycerol solution in a screw-top cryotube.
3. Vortex briefly.
4. Incubate at room temperature for 1/2 hour, and place directly into a –80°C freezer.
5. Prior to freezing, label the tube with the Biobrick part number, plasmid, and antibiotic resistance.

Electro-transformation

Materials

• LB broth
• plasmid DNA
• cuvettes
• Gene Pulse apparatus
• incubator at 37°C
• LB plates with appropriate antibiotic

Procedure

1. Prepare eppendorf tubes with 1 ml LB medium.
2. Gently thaw the cells at room temperature and place them on ice.
3. In a cold, 1,5ml polypropylene tube, mix 100 µl of the cell suspension with 1 to 2 µl of DNA (DNA should be in a low ionic strength buffer such as TE). Mix well and let sit on ice for 0,5 to 1 minute.
4. Set the Gene Pulser apparatus at 25 µF and 2,5 kV. Set the Pulse controller to 200 Ohm.
5. Transfer the mixture of cells and DNA to a cold, 0,2 cm electroporation cuvette (yellow), and shake the suspension to the bottom of the cuvette. Place the cuvette in a chilled safety chamber slide. Puysh the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber.
6. Pulse once at the above settings. This should produce a pulse with a time constant of 4 to 5 msec. (The field strength will be 12,5 kV/cm.)
7. Remove the cuvette from the chamber and immediately add 1ml of LB medium to the cuvette and quickly resuspend the cells with a pasteur pipette. (This rapid addition of LB after the pulse is very important in maximizing the recovery of transformants.)
8. Transfer the cell suspension to a 17 x 100mm polypropylene tube and incubate at 37°C for 1 hour. (Shaking the tubes at 225 RPM during this incubation may improve the recovery of transformants.)
9. Plate on selective medium.

Taq PCR

Mix

Prepare this mix sterile and on ice.

1. 2,5µl dNTP's (2mM)
2. 1,25µl Primer$_1$ (20$\mu$M) $+$ 1,25$\mu$l Primer$_2$ (20$\mu$M)
3. 2µl Template
4. 2,5µl Taq buffer
5. 0,2µl Taq DNA polymerase
6. 15,3µl MQ

Cycles

1. 1 x 6min 94°C
2. 30--35 x
   • 1min 94°C
   • 30sec 55-60°C (lowest Tm minus 2°C)
   • 1min/kb 72°C
3. 1 x 6min 72°C
4. stay 4°C

Pfx PCR

Mix

Prepare this mix sterile and on ice. Afterwards, put in 4 eppendorf tubes 50$\mu$l of this mix.

1. 40 µl Amplification Buffer (blue)
2. 6 µl dNTP's (10mM)
3. 4 µl {MgSO_4} (white)
4. 3 µl Primer1 (20µM) + 3µl Primer2 (20µM)
5. 4 µl Template
6. 3,2 µl Pfx DNA polymerase (black)
7. 40 µl Enhancer (red)
8. 96,8 µl MQ (very sterile)

Cycles

1. 1 x 6min 94°C
2. 30--35 x
   • 1min 94°C
   • 30sec 55-60°C (lowest Tm minus 2°C)
   • 1min/kb 72°C
3. 1 x 6min 72°C
4. stay 4°C

Ligation

Materials

• vector + insert DNA (digested)
• microcentrifuge tubes
• heat block (45°C)
• Buffer for ligation
• T4 DNA ligase
• Deionized, sterile water

Procedure

1. Add 50 ng vector to x ng insert to a microcentrifuge tube. Calculate x using the following formula: bp V/ bp I = 50 ng V / x ng I
2. Put the tube in a heat blok at 45°C for 5 minutes.
3. Add 2 µl Ligation Buffer
4. Add 1 µl T4 DNA ligase (keep on ice - very sensitive to temperature)
5. Add sterile water untill you have 10 µl (if necessary).
6. Incubate them overnight at 16°C.

PCR with end filling

Materials

• primers
• Klenow buffer
• mQ
• dNTP
• Klenow polymerase

Procedure

1. Make 6 PCR tubes with 2 µl primer 1 (1 µM), 2 µl primer 2 (1 µM), 2µl klenow buffer and 11µl mQ
2. 3’ at 95°C
3. 5’ at (annealingt° overlap)
4. Add 2µl dNTP (2µM) and 1µl Klenow polymerase
5. 30’ at 37°C
6. 20’ at 75°C
7. Do a PCR to amplificate

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