Team:Northern BC/Experiments
Northern BC iGEM 2019 Protocols
Site-Directed Mutagenesis
Primer Design:
Design primers that are 30 - 45 base pairs in length, depending on the number of bases to be changed. Centering the primers over the position to be changed works best, however the primers could be offset to avoid formation of primer dimers. We haven’t found this to be a problem and tend to center our primers.
It is helpful to introduce a restriction site, if possible, so colonies can be screened by restriction enzyme digest rather than by sequencing. This saves time and money, although the final candidate will still have to be sequenced to confirm the correct mutation has been made without the introduction of additional mutations. The restriction enzyme site would have to be unique; that is, not present anywhere else in the plasmid.
Template Plasmid:
Smaller plasmids are generally easier to work with, although we have had success with larger plasmids (~ 8 kbp). Plasmids such as pUC18 or pUC19 work well for site-directed mutagenesis. Mini-prep the template plasmid from an overnight liquid culture.
PCR Reaction and Conditions:
Add the following components in this order. Keep the DNA Polymerase on ice at all times.
A different DNA Polymerase may be used, but it must be high fidelity to avoid introducing random mutations. Taq DNA Polymerase should not be used for this purpose. We have been using Phusion® High-Fidelity DNA Polymerase (NEB Cat# M0530).
Program:
Stage 1: 98 °C for two minutes
Stage 2: 98 °C 10 seconds (denature)
54 °C 45 seconds (primer annealing)
72 °C 10 minutes (extension)
*The extension temperature should be set according to the manufacturer’s recommendations for your DNA polymerase
Repeat stage two for 18 cycles
Stage 3: 72 °C 20 minutes (final extension to catch any strands that were not fully extended earlier)
Stage 4: 4 °C indefinitely
Setting the program to end with an indefinite 4 °C period allows one to leave the reaction run over night. Leaving it at 4 °C in the thermocycler is no different from leaving it in the fridge. Running the PCR overnight means the reaction will be ready to work with first thing the following morning.
DpnI Digest:
There is now mixture of plasmid carrying the mutation and the original parental wild type DNA, so the parental DNA will need to be removed. Since bacteria methylate their DNA, the parental DNA will be methylated while all PCR-generated DNA will not. DpnI is a restriction enzyme that recognizes and cleaves a consensus sequence only when the DNA is methylated. Thus treatment with DpnI will degrade all wild type DNA, leaving only the PCR-generated mutant.
Add 1 uL of DpnI (NEB Cat# R0176S) directly to the reaction and incubate at 37 °C for at least two hours, but not more than 16 hours. We generally find three to four hours to be sufficient.
Precipitate DNA to Concentrate and Clean-up:
To your PCR reaction add 50 uL dH2O, 5 uL 5 M NaCl,1 uL 20 mg/mL glycogen, and 1 mL cold 100% ethanol.
Spin in a microcentrifuge at 13,000 rpm at 4 °C for at least 10 minutes, but not more than 30 minutes. Ideally, DNA should pellet at the tip of the tube, but it should not be packed so tight that it is hard to resuspend.
Pour off the ethanol and gently add 1 mL cold 70% ethanol to wash the pellet (do not resuspend the pellet).
Spin again as above, but for only five minutes.
Remove all ethanol and air dry on the bench for approximately 10 - 15 minutes. Do not let this go for too long as a pellet that is over dried seems to be harder to resuspend, affecting the transformation efficiency.
Transform the whole reaction into E. coli (DH5𝝰). We tend to resuspend the DNA with 50 uL of cells and carry on directly with the transformation protocol, but it could instead be resuspended in 5 uL of dH2O and then added directly to the cells.
Troubleshooting:
Presence of wildtype transformants this could be because the parental DNA is not efficiently methylated. Grow the cells in which the plasmid has been propagated for 24 hours to make sure the DNA is fully methylated before your mini-prep. You could also try using less template in the PCR or increasing the number of cycles in stage 2 of the program to 30.
Bacterial Transformation
Add 5 - 10 uL of ligation reaction or up to 25 ng of circular plasmid DNA to 50 uL of RbCl2 competent cells, making an effort not to use a volume exceeding 5% of the volume of cells used.
Note: Use DH5𝝰 for plasmid storage and/or propagation and BL21, Rosetta, or other expression strain for protein expression and purification from a bacterial host.
Controls: Set up a negative and a positive control to assess cell competency
(+) Control: 1 - 10 ng of supercoiled plasmid DNA (pUC19 works well) expressing the appropriate selective marker
This should generate a ‘lawn’ of colonies - too many to count
(-) Control: cells with no DNA
Since the reactions will be plated on selective media, any cells that have not taken up the plasmid conferring resistance to
the chosen antibiotic will not survive. Thus, there should be no growth on this plate.
Store all tubes on ice for 30 minutes.
Heat shock each reaction in a water bath for 90 seconds at 42 °C. The cells must be brought to temperature at an appropriate rate; thus, a water bath is most suitable for this purpose. The length of incubation is a function of the thickness of the microcentrifuge tube wall. If you find your transformations are not working well you can vary the incubation time. Most protocols call for an incubation time of anywhere from 30 seconds to two minutes, but 90 seconds has always worked well for us.
Without shaking the tubes, gently transfer them directly to ice and allow the cells to cool for two minutes.
Add 100 uL of liquid LB (no antibiotic) and incubate on a gently shaking orbital shaker for 30 - 45 minutes at 37 °C. If a shaker is not available place the tubes in a 37 °C incubator or water bath and periodically and gently agitate them. This is a recovery phase in which the cells are healing after heat shock. You must be gentle with them and provide enough time for them to begin producing the antibiotic resistance selective marker expressed from the plasmid before plating on selective media.
Working near a flame and using aseptic techniques, plate the entire 100 uL transformation onto a selective plate. We like to pre-incubate our plates at 37 °C to avoid shocking the cells again. Pipette the aliquot to the center of the plate and then add four to five sterile glass plating beads. Cover the plate with the petri dish lid and gentle roll the beads to spread the cells over the entire surface of the plate. Pour the used beads into a weak bleach solution for cleaning, sterilization and re-use.
Incubate plates overnight (approximately 16 hours) at 37 °C. Do not leave plates longer than 16 - 20 hours. Plates can be stored at 4 °C until one is ready to work with a colony.
