Materials

The restriction enzymes used for digestion were provided by the company Fermentas. These restriction enzymes belong to the restriction enzymes line: FastDigest. (http://www.fermentas.com/en/products/all/fastdigest-restriction-enzymes) The enzymes used for PCR were taq polymerase, dream taq polymerase and pfu polymerase and the enzyme used for ligation is T4 DNA ligase. These enzymes were also provided by Fermentas.
DNA purification was done with the High Pure PCR Purification Kit provided by Roche. The plasmid preps were done with the High Pure Plasmid Isolation Kit also provided by Roche.
For analysis of PCR products, the following DNA ladders were used: Generuler 1kb DNA ladder and Generuler 100bp DNA ladder provided by Fermentas.
Thermocyclers used for PCR and digestion: VWR™, serial number: 1200-10906 and 1200-11337

Cloning strategy

Digestion and ligation
Making competent cells and transformation protocol
Antibiotics
Colony PCR

To clone a desired DNA fragment into the vector, a PCR with Pfu polymerase needs to be done. You can find the protocol for PCR with Pfu polymerase on: http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_ep0501.pdf
Plasmids were obtained with the help of the High Pure Plasmid Isolation Kit provided by Roche.

Digestion and ligation

150 ng of the vector needed to be digested. To calculate how much ng insert we need to use for the ratio vector: insert being 1:6, the Ligation calculator was used: http://www.insilico.uni-duesseldorf.de/Lig_Input.html.
The amount of DNA was digested in a total volume of 20μl for the digestion of the insert and 30μl for the digestion of the vector, since FastAP was used for reducing self ligation. Otherwise, the glycerol concentration would be too high because of the addition of FastAP to the digestion mixture. 1μl of the restriction enzyme was used in each reaction. Digestion was done at 37 degrees and for 30 to 60 minutes.
After digestion, the DNA was purified with the help of the High Pure PCR Purification Kit provided by Roche. Afterwards, the ligation was done in a total volume of 20μl containing 1μl T4 DNA ligase, 2μl T4 DNA ligase buffer and the digested+purified insert and vector and, if necessary, the additional MQ water. Ligation was done at room temperature for 30 to 40 minutes.

Back to cloning strategy

Making competent cells and transformation protocol

Competent DH5alpha cells were made and transformed with the following adapted protocol of Robyn Eilander (MolGen Groningen):
Materials to be supplied by the user

• 1.5 ml microcentrifuge tubes (sterile, dry, 4 °C, min. 200 pieces)
• 50 ml plastic centrifuge tubes (sterile, pre-cooled, 8 pieces)
• cuvettes
• 2 L flask (sterile, 2 flasks)-preferably Erlenmeyers
• stove, 37 °C
• TY broth (sterile, 5 ml)
• 2xTY broth (sterile, 37 °C, 500 ml)
• TY agar plate
• E. coli strain from glycerol stock
• Buffer RF1 (ice cold)
• Buffer RF2
• ice
• liquid nitrogen
Buffer RF1 500mls
Rubidium chloride 6.00 g
Potassium acetate 2.45 g
Calcium chloride 2H20 0.75 g
Glycerol 75.00 g
Adjust to pH 5.8 using 0.2 M Acetic acid
Sterilise by filtration
Buffer RF2 300mls
0.5 M MOPS PH 6.8 6.00 ml (Use 4 M NaOH to pH MOPS)
Rubidium chloride 0.36 g
Calcium chloride 2H20 3.30 g
Glycerol 45.00 g
Adjust to pH 6.8 with 1M NaOH
Sterilise by filtration


Protocol
Day 1
1. Streak out E. coli strain, direct from a glycerol stock 24 hours before you intend to put up an overnight
culture.
2. Autoclave 200 1.5 ml microcentrifuge tubes and ensure they are dry before use. (Leave the dry tubes at 4 °C so
they are pre-cooled before use.)
Day 2
1. ~5pm day before growing cultures, inoculate 5 ml 2xTY with a freshly grown colony. Grow O/N at 37 °C.
2. It is a good idea to put 2x 2 litre sterile flasks & 500 ml sterile 2xTY at 37 °C overnight – this will create
continuity of temperature when you inoculate your 2x 200 ml cultures the next morning.
Day 3
1. Add 200 ml pre-warmed 2xTY into a 2 litre flask. Inoculate with 2 ml (1/100 dilution) of the overnight
culture. Grow 2x 200 ml cultures at the same time. Grow cultures to an OD600nm of between 0.3-0.4.This should
take a maximum of 2 hours.
2. Put 8x 50 ml plastic centrifuge tubes in ice to pre-cool. Make sure your centrifuge has been pre-run to bring
down the temperature to 4 °C.
3. Collect culture into 8 x 50ml pre-cooled centrifuge tubes. Leave on ice for 15 minutes.
4. Pellet the cells by centrifuging at 2700 rpm for 15 minutes 4 °C.
5. Remove the supernatant. Drain residue supernatant from the cell pellets. Do this by turning the tubes upside
down on paper towels. Remove the supernatant left on the tube lip and inside wall using tissue. Put the tubes
back on ice.
6. Resuspend cell pellets by adding 16 ml of ice cold RF1 to each pellet and vortexing gently. (Do this just
enough to resuspend the cells quickly). Return the tubes to ice for 15 minutes.
7. Pellet the cells by centrifuging at 2700 rpm for 15 minutes 4 °C.
8. Remove the supernatant. Drain residue supernatant from the cell pellets. Do this by turning the tubes upside
down on paper towels. Remove the supernatant left on the tube lip and inside wall using tissue. Put the tubes
back on ice.
9. Add 4 ml of RF2 to each cell pellet. Gently resuspend each pellet. Leave the resuspended pellets on ice for 15
minutes.
10. Get some liquid nitrogen.
11. Dispense 4 ml of resuspended cell pellet in aliquots of 200 μl to each 1.5 ml microcentrifuge tubes (can use
a dispenser pipette). After aliquoting 4 ml from one tube shut the lids on the 1.5 ml tubes and flash freeze in
liquid Nitrogen. Repeat this process with all 8 resuspended cell pellets.
Store the frozen aliquots of competent cells at -80 °C.

For transformation

• Add DNA (ligation mixture or plasmid) to 40ul of cells from -80C stock
• 30 min on ice
• Heat shock: 45 seconds @ 42C
• Tubes back on ice and add 300 ul TY medium
• 45 min – 1,5h at 37C
• Plate 25 and 250 ul on TY-agar plates containing antibiotic
________________________________________
NOTE (by Robyn): 2xTY broth (apparently required for the preparation of the competent cells) can also be replaced by simple TY broth. So don't bother preparing 2xTY! Protocol works brilliantly.

Back to cloning strategy

Antibiotics

Ampicillin
100 mg/ml Ampicillin (1000x) Stock

1 g of Ampicillin sodium salt in 10 mL of demiwater (or 50% EtOH)
Add NaOH or KOH to allow the Ampicillin to dissolve
Filter sterilize 0.2 μm filter and aliquot
Store -20 °C
http://openwetware.org/wiki/Ampicillin

Chloramphenicol
35 mg/ml Chloramphenicol (1000x) Stock

0.35 g in 10 mL 100% EtOH
Filter sterilize 0.2 μm filter and aliquot
Store -20 °C
http://openwetware.org/wiki/Chloramphenicol

Kanamycin
50 mg/ml Kanamycin (1000x) Stock

500 mg in 10 mL demi water
Filter sterilize 0.2 μm filter and aliquot
Store -20 °C
http://openwetware.org/wiki/Kanamycin

Back to cloning strategy

Colony PCR

To test whether the colonies on your plates contain the right insert, a colony PCR needs to be done.
Colony PCR per reaction in total volume of 20μl:
Taq 10× buffer: 2μl
dNTPs 10mM: 0.4μl
MgCl2: 1.2μl
Forward Biobrick primer: 0.4μl
Reverse Biobrick primer: 0.4μl
Taq DNA polymerase: 0.1μl
MQ water: 15.5μl

PCR conditions:
Preheated lid: 111°C
Denaturation: 94°C for 10 min.
Cycle 33×:
denaturation: 94°C for 30s.
annealing: 60°C for 30s.
Extension: 72°C for ... min (speed of taq: 1kb/min.
Final extension: 72°C for 10 min.
Store infinite at 4°C

Analyse on a 1% TBE agarose gel.
Back to cloning strategy