Bunsen required for all steps.
1.Label tube with the name of the plasmid being added.
2.Locate DNA on kit plate (see iGEM distribution catalog).
3.Pierce the foil with a pipette containing 10 μl of sterilized water.
4.Mix well by taking up and releasing mixture multiple times.
5.Place the suspended DNA solution in a 1.5 ml Eppendorf, leaving on ice.
Team:Warwick/Protocols
USING iGEM DISTRIBUTION KIT
TRANSFORMATION
1.Collect electrocompetent cells from – 80 freezer and keep on ice.
2.Add 1 μl of plasmid to tube containing 55 μl of competent cells.
3.Place electroporation cuvettes on ice to chill.
4.Mix 56 μl mixture and transfer to chilled electroporation cuvettes, ensuring mixture is released between the two metal plates.
5.Ready 950 μl of SOC in a pipette.
6.Place the electroporation cuvette into the electroporator.
7.Make sure the electroporator is set to bacteria.
8.Slide electroporation cuvette into position.
9.Press pulse.
10.Immediately remove cuvette and lid, and add the 950 μl SOC.
11.Place cuvette on ice.
12.Transfer contents of cuvette back into Eppendorf (using 950 pipette) and label with the name of the plasmid.
13.Repeat for however many plasmids you wish to transform.
14.Allow cells to recover for 1 hour at 37 °C with the SOC.
15.Allow cells to grow on plates with appropriate antibiotic(s).
INNOCULATION
1.Setup and maintain sterile techniques through out.
2.Prepare the 15 ml falcon tubes, labelling appropriately (antibiotic and date).
3.Place 5 ml of LB in each tube.
4.Add the appropriate concentration of antibiotic into the falcon tube (1000x dilution).
5.If this is a colony inoculation, touch the colony with a pipette tip and place into the falcon tube.
6.If this is a glycerol stock inoculation then scrape the surface with a pipette tip and mix contents into the falcon tube.
7.Place in a shaking incubator at 37 °C for 16 hours and store at 4 °C ready for mini-prep or making a glycerol stock, depending on the type of inoculation.
MAKING A GLYCEROL OF STOCK CELLS
Bunsen required for all steps.
1.Vortex inoculated bacteria containing the desired plasmids (in LB).
2.Add 60 μl of inoculated cells into a 1.5 ml Eppendorf, followed by 40 μl of 50% glycerol.
3.Vortex the new 20% glycerol solutions and leave in - 80 oC freezer.
MAKING A 1% AGAROSE GEL
1. 2.5g agarose + 250 mL 1xTAE buffer (for 1% agarose).
2. Mix and heat in microwave ~3 mins with stirring till clear with no bits.
3. Assemble gasket and combs.
4. Add ~50 ml agarose solution to gasket to just below the tops of the combs.
5.Store remaining agarose at 60 °C in oven.
6. Add 5 μl cybrsafe (in draw by sink, store in dark).
7. Leave to set for 20 minutes.
8. Remove rubber dams carefully to avoid tearing the gel.
9. Place gasket into tank with combs furthest from you at the black end.
10. Fill with 1 x TAE buffer to just below max fill line.
11. Remove combs to leave wells.
MAKING AND RUNNING A GEL
Sample preparation
1. Bring out loading dye and 1 kB ladder from freezer and allow to thaw.
2. Add 1/5th loading dye by volume to each sample (e.g. 10 μl loading dye to 50 μl of sample).
3. Vortex samples briefly to mix.
Loading Samples
1.Add 6 µl of 1 kB ladder to first well + later wells if running a lot of samples
2. Add up to 30 µl of sample to other wells
Running gels
1. Connect the lid to the top of the tank with the black clip in the black hole and the red clip in the red.
2. Run at 90 V for 30 mins as standard
3. Run time can be adjusted by pressing mode button 3 times and using up and down arrows.
Example:- Running a gel of PCR products
Sample sizes = 50 µl
Number of samples = 4
1.Add 10 µL of loading dye to each sample
2.Loading 30 µl of sample into gel as shown below.
GEL EXTRACTION
1. Using a QIAquick gel extraction kit, add ethanol to buffer PE
2. Slice the DNA fragment from the agarose gel with a scalpel and weigh
3. Add 3 volumes of buffer QG to 1 volume of gel and incubate, at 50 °C, for 10 min (vortex every 2-3 min to help the gel dissolve)
4. If the solution is orange or violet add 10 µl of 3 M sodium acetate, pH 5.0, to turn the mixture yellow
5. Add 1 gel volume of isopropanol and mix
6. Place a QIAquick column in a 2 ml collection tube and add the sample
7. Centrifuge for 1 min and discard the flow-through
8. For DNA sequencing, vitro transcription or microinjection – add 500 µl of buffer QG, centrifuge for 1 min and discard the flow-through
9. Wash the sample by adding 750 µl of buffer PE (allow to stand for 2-5 min if it is being used for salt-sensitive applications), centrifuge for 1 min and discard the flow-through.
10. Place the QIAquick column into a clean 1.5 mL microcentrifuge tube and elute the sample in 20 µL of buffer EB, making sure the buffer is added to the centre of the membrane. Do not touch the membrane with the pipette tip.
11. Allow the sample to stand for 1 min and then centrifuge for 1 min, collecting the flow-through
PCR REACTION AND PURIFICATION
1. Using a QIAquick PCR purification kit, add ethanol to buffer PE and 1:250 volume pH indicator 1 to buffer PB
2. Add 5 volumes of buffer PB to 1 volume of PCR reaction, adding 10 µl of 3 M sodium acetate (pH 5.0) if the solution is orange or violet to turn it yellow
3. Place the QIAquick column into a 2 ml collection tube, add the sample and centrifuge for 1 min, discarding the flow-through
4. Add 750 µl of buffer PE, centrifuge for 1 min, discard the flow-through and centrifuge for a further 1 min
5. Place the QIAquick column into a 1.5 ml microcentrifuge tube
6. Elute the DNA with 50 µl of buffer EB or water (20 µl for a higher concentration), making sure it is added to the centre of the membrane, and allow to stand for 1 min
7. Centrifuge for 1 min and collect the flow-through
MINIPREP
1. Using a QIAquick spin miniprep kit, add ethanol to buffer PE
2. Pellet a 1-5 ml culture grown overnight by centrifuging at 8000 rpm for 3 min, at room temperature
3. Discard the supernatant, re-suspend the pellet in 250 µl of buffer P1 and transfer the mixture into a microcentrifuge tube
4. Add 250 µl of buffer P2, invert the tube 4-6 times until the mixture is clear, and do NOT allow the lysis solution to proceed for more than 5 min
5. Add 350 µl of buffer N3, invert the tube 4-6 times and centrifuge for 10 min
6. Place 800 µl of the supernatant from step 5 into a QIAprep 2.0 spin column, centrifuge for 1 min and discard the flow-through
7. Wash the QIAprep 2.0 spin column with 0.5 ml of buffer PB, centrifuge for 1 min and discard the flow-through
8. Wash again with 0.75 ml of buffer PE, centrifuge for 1 min, discard the flow-through and centrifuge for a further 1 min
9. Place the QIAprep 2.0 spin column into a 1.5 ml microcentrifuge tube and elute, making sure the liquid is placed on the centre of the membrane, with 30 µl of buffer EB (10 nM TrisCl, pH 8.5) or water
10. Allow the column to stand for up to 4 mins and centrifuge for 1 min, collecting the flow-through
11. Transfer flow-through to a new 1.5 ml microcentrifuge tube and label accordingly.
NANODROP
1. Click on Nucleic Acids
2. Add 1 µl water. Close and re-open the lid, then wipe it clean.
3. Add 1 µl Buffer EB (or water, depending on what the sample is eluted in), and blank the machine.
4. Add 1 µl of the first sample and test. Make note of the nucleic acid concentration (ng/µl) on the side of the microcentrifuge tube.
5. Repeat for all samples.
6. Test with buffer EB to check if the machine is still calibrated properly.
DIGESTION
1. Prepare the following in separate PCR tubes for all samples requiring digestion:
The restriction enzyme used is dependent on the cut sites present on the template DNA and the purpose of the digestion. Triple digestions can be completed with an additional restriction enzyme if necessary. When used for diagnostic purposes, the solution is made up to 20 µl instead of 50 µL.
This is then incubated in a thermocycler at 37 °C for one hour (the samples can be incubated for longer depending on the star activity of the restriction enzymes).
PCR (PHUSION)
1. Assemble all reaction components in the following table on ice. Making a master mix may be useful when using very small volumes of the components.
2. Thermocycling conditions for a routine PCR:
COLONY PCR
1. Pick up a colony from a plate and resuspend in 100 µl of water. Use this as the template.
2. Assemble all reaction components in the following table on ice. Making a master mix may be useful when using very small volumes of the components.
3. Thermocycling conditions for a routine colony PCR:
LIGATION
1. Prepare the following in separate PCR tubes for all samples requiring ligation:
2. Incubate at 16 °C for 16 hours and then store at 4 °C
GROWING COMPETENT CELLS
Main step 1: Growing electrocompetent cells
1. Add 200 ml LB media into a 1 l Erlenmeyer flask.
2. Add 200 µl antibiotic into the Erlenmeyer flask.
3. Measure the optical density (OD) of cells grown overnight in the 15 ml Falcon tube. OD of 1 for E. coli means 108 number of cells.
4. We want to have OD of 0.01 in the Erlenmeyer flask at the start of the experiment.
5. Place the Erlenmeyer flask in the incubator at 37 °C with 300 RPM (cover the opening of the flask with foil).
6. Be very careful of the cells OD in the Erlenmeyer flask. The best OD range for the electrocompetent cell is 0.4 to 0.6. Therefore, at least 2 times sampling and measuring cells OD is necessary to monitor the exponential increase in the cells OD. It usually takes ~3 hours for electrocompetent cells growing at 37 °C to reach OD of 0.5.
Main step 2: Harvesting electrocompetent cells
1. Turn on the centrifuge and keep its temperature cool at 4 °C.
2. Take the cells out of the incubator when OD reaches 0.4-0.6 (caution needed).
3. Transfer the electrocompetent cells to 4 x 50 ml Falcon tubes while keeping the Falcon tubes in ice water.
4. Place the Falcon tubes in the centrifuge and spin down (4000 RPM, 15 mins, 4 °C).
5. Pour out supernatant quickly and be careful of cell pellets.
6. While keeping the tubes in ice water, gently resuspend (DO NOT VORTEX) the cell pellets in each Falcon tube with 25 ml 10% glycerol.
7. Transfer the 4 x 25 ml of solution from the 4 x 50 ml Falcon tubes into 2 x 50 ml Falcon tubes (keep them cold).
8. Place the Falcon tubes in the centrifuge and spin down again (4000 RPM, 15 min, 4 °C).
9. Pour out supernatant quickly and be careful of cell pellets.
10. While keeping the tubes in ice water, gently resuspend the cell pellets in each Falcon tube using 25 ml 10% glycerol.
11. Transfer the 2 x 25 ml of solution from the 2 x 50 ml Falcon tubes into the 1 x 50 ml Falcon tube (keep it cold).
12. Place the Falcon tube in the centrifuge, along with a balance tube of water, and spin down again (4000 RPM, 15 min, 4 °C).
13. Pour out the supernatant quickly and be careful of cell pellets.
14. While keeping tubes in ice water, gently resuspend the cell pellets in the Falcon tube in 2 ml of 20% glycerol.
15. In the cold room, aliquot ~55 µl of cells into as many 1.5 ml microcentrifuge tubes as possible and as fast as possible.
GOLDENGATE ASSEMBLY
1. Prepare the following in separate PCR tubes for the components that need to be assembled: (more than one insert can be added to the reaction mixture)
2. Thermocycling conditions for a goldengate assembly reaction:
3 min at 37 °C
4 min at 16 °C
(25 cycles)
5 min at 50 °C
20 min at 80 °C
∞ at 4 °C
3. This is now ready to be transformed into competent cells and/or run a diagnostic gel to check for successful assembly.
GIBSON ASSEMBLY
1. Prepare the following in separate PCR tubes for the desired samples:
2. Incubate for 1 hour at 50 °C.
3. This is now ready to be transformed into competent cells and/or run a diagnostic gel to check for successful assembly.
