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Team:Groningen/Template/MODULE/Notebook/protocols
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Latest revision as of 03:01, 18 October 2014
Notebook
>
Protocols
Polymerase Chain Reaction
All PCR reactions were done with DreamTaq and Phusion polymerase from Thermo Scientific.
DreamTaq polymerase
For colony PCR DreamTaq polymerase is used. As the template, the colony of interest is picked from a plate using a sterile toothpick and mixed into the reaction mixture. The cycling conditions are listed in the table below.
| Step | Temperature | Time | Number of cycles |
|---|---|---|---|
| Initial denaturation | 95 °C | 1 - 3 min | 1 |
| Denaturation | 95 °C | 30 sec | 30 |
| Annealing | Tm - 5 °C | 30 sec | |
| Extension | 72 °C | 1 min/kb | |
| Final extension | 72 °C | 10 min/kb | 1 |
Phusion polymerase
Phusion polymerase is used when high fidelity is needed for the reaction. Phusion has been used for making of the BioBricks with PCR and generating specific mutations in genes with the use of primers. The cycling program is listed in the table below.
| Step | Temperature | Time | Number of cycles |
|---|---|---|---|
| Initial denaturation | 98 °C | 30 sec | 1 |
| Denaturation | 98 °C | 10 sec | 30 |
| Annealing | Tm - 5 °C | 30 sec | |
| Extension | 72 °C | 15-30 sec/kb | |
| Final extension | 72 °C | 10 min/kb | 1 |
With difficult templates GC buffer with 1.5% DMSO was used.
Transformation of Escherichia coli
Buffers
500 ml RF1 buffer
Adjust the pH to 5.8 using 0.2 M acetic acid. Sterilise by filtration.
| Component | Amount | Rubidium chloride | 6.00 g |
|---|---|
| Potassium acetate | 2.45 g |
| Calcium chloride * 2 H2O | 0.75 g |
| Glycerol | 75.00 g |
300 ml RF2 buffer
Adjust the pH to 6.8 with 1 M NaOH. Sterilise by filtration.
| Component | Amount |
|---|---|
| 0.5 MOPS pH 6.8 | 6.00 ml |
| Rubidium chloride | 0.36 g |
| Calcium chloride * 2 H2O | 3.30 g |
| Glycerol | 45.00 g |
Preparing chemically competent E. coli
Grow an overnight culture E. coli in 5 ml LB, at 37 °C. Inoculate 200 ml of prewarmed LB with 2 ml of the overnight culture the next morning. Grow the cultures until an OD600 of 0.3 - 0.4 is obtained. Put the cells on ice for 15 minutes and then pellet them by centrifuging them for 15 minutes with 2700 rpm at 4 °C. Resuspend the pellets in 16 ml RF1 buffer and leave them on ice for 15 minutes. Centrifuge the cells again for 15 minutes with 2700 rpm at 4 °C. Resuspend the pellet in 4 ml RF2 buffer. Flash freeze aliquots of the cells using liquid nitrogen.
Transformation
Add the DNA to 50 μl chemically competent E. coli. Put the cells on ice for 30 minutes. Give the cells a heatshock by placing them at 42 °C for 45 seconds. Put the tubes back on ice and add 300 ml LB medium. Incubate the cells for an hour at 37 °C. Plate the cells on LB agar.
Restriction
For restriction FastDigest enzymes from Thermo Scientific were used. For digestion of up to 1 μg DNA, 0.8 μl enzyme is used. The reaction is incubated at 37 °C for 30 minutes. The enzymes are then deactivated by cleaning the restrictionmixture using the PCR Purification Kit from Thermo Scientific or by incubating the mixture at 80 °C for 20 minutes.
Ligation
For ligation of DNA fragments 1U T4 DNA ligase was used. The amount of vector DNA that is used is 20 - 100 ng. The amount of insert in ng can be calculated using (ng of vector * kb of insert)/kb of vector * R with R being the molar ratio of the insert over the vector. The mixture is incubated for 1 hour at roomtemperature. After incubation, 5 μl of the mixture can be used for the transformation of 50 μl chemically competent cells or 1 μl for electrocompetent cells.
Gibson assembly
Gibson assembly is a nice way to join multiple DNA fragments without creating a scar between the fragments, see figure 1. For the assembly primers are designed in such a way that all the fragments have a 40 bp overlap at their ligation point. Then, a PCR is performed on all the fragments to create the overhang (1). Finally, the assembly is done. The assembly uses a T5 exonuclease to create single-stranded 3' overhangs that cause annealing of complementary fragments. (2) Then a polymerase in the same mixture fills in the gaps between each fragment and a DNA ligase seals the nicks. (3) The ligated fragments can then directly be used to transform the bacterium of your choice. (4)
Figure 1:
Gibson assembly
Procedure
A PCR is done on the fragments with the primers that were designed for the assembly. Then the assembly is performed in the 2x Gibson Assembly Master Mix from NEB. The fragments are added to 10 μl of this mix, 0.25 pmol each, giving a total volume of 20 μl. The mix is then incubated at 50 °C for 1 hour.
Transformation of Lactococcus lactis
Preparing electrocompetent L. lactis
An overnight culture of L. lactis is made in SMGG. The following day, 10 ml of the overnight culture is pipetted in 100 ml SMGG and grown at 30 °C until an OD600 of 0.2 - 0.7 is reached. The cells are then three times washed with 50 ml ice cold wash buffer (0.5 M sucrose and 10% glycerol). The cells are then resuspended in 1 ml wash buffer and can be used directly or stored at -80 °C.
Electroporation
For the electroporation 1 μl of the DNA is added to 40 μl cells in an ice cold cuvette. The cuvette is then electroporated at 2.5 kV, 25 μF and 200 Ω. Then, 1 ml SMG17MC is added and the cells are incubated at 30 °C for two hours. The cells can then be plated on GSM17-agar plates.
