PCR (Phusion polymerase)

1. Prepare the mix for all the samples in a single 1.5mL tube (mind pipetting error!). For one sample:

   Component	Volume (µL) for 20µL reaction	Volume (µL) for 50µL reaction
   5X Phusion HF Buffer	4	10
   10mM dNTPs	0.4	1
   FW Primer	1	2.5
   RV Primer	1	2.5
   Phusion polymerase	0.2	0.5
   Template DNA (<250ng)	Variable	Variable
   Nuclease-free H2O	Up to 20µL	Up to 50µL

   NOTE: The 250ng of template DNA are approximate, choose a volume that works fine for all your samples and fill it in the excel sheet so you are able to prepare a mix for all samples at the same time.
2. Add the total volume per sample (without DNA) to each PCR tube.
3. Add the template DNA to each tube.
4. Put the tubes in the PCR machine and apply the following program (it needs to be adjusted for primers annealing temperature and extension time):

   Step	Temperature (ºC)	Time
   Initial denaturation	98	30sec
   Denaturation	98	10sec
   Annealing	Tm-3	20sec	x30 cycles
   Extension	72	15-30sec/1kb
   Final extension	72	5min
   	4	Hold

5. Run a DNA electrophoresis following the DNA electrophoresis protocol.

Gel isolation

1. Run a DNA electrophoresis (following the DNA electrophoresis protocol), loading the full volume of PCR or restriction product into the agarose gel. If necessary, use multiple wells.
2. Cut the band (or bands) corresponding to the fragment of interest with a razor blade and put it in a pre-weighted 1.5mL tube, then calculate the weight of the gel fragment.
3. Add 10µL of Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 50-65ºC until gel slice is completely dissolved.
4. Pre-warm an aliquot of Nuclease-free water at 50-65ºC.
5. Insert SV Minicolumn into Collection Tube and label both of them according to the labelling of your samples.
6. Transfer the dissolved gel mixture to the Minicolumn assembly. Incubate at room temperature for 1 minute.
   NOTE: When pipetting into the column, aim the pipette to the wall not the membrane to avoid damaging it.
7. Centrifuge the SV Minicolumn assembly at maximum speed for 1 minute.
8. Discard the flowthrough and reinsert the SV Minicolumn into the Collection Tube.
9. Add 700µL of Membrane Wash Solution (if it is the first use, dilute it with 95% ethanol following the bottle’s instructions).
10. Centrifuge the SV Minicolumn assembly at maximum speed for 1 minute.
11. Discard the flowthrough and reinsert the SV Minicolumn into the Collection Tube.
12. Repeat steps 9-11 with 500µL of Membrane Wash Solution and centrifuging for 5 minutes.
13. Once the Collection Tube is empty, centrifuge the Minicolumn assembly at maximum speed for 1 minute with the microcentrifuge lid open to allow ethanol full evaporation.
14. Transfer the SV Minicolumn to an empty 1.5mL tube carefully labelled.
15. Add 500µL of the pre-warmed water (300µL for higher concentrations or when small amounts of DNA are suspected) directly to the centre of the SV Minicolumn, without touching the membrane with the pipette tip.
16. Incubate at room temperature for 5 minutes.
17. Centrifuge at maximum speed for 1 minute.
18. Discard the SV Minicolumn, cap the tube containing the eluted DNA and keep at 4ºC (for immediate use) or -20ºC (for storage).

Restriction

1. Get the detailed protocol for restriction from the following website, according to the enzymes used. When high restriction product concentrations are needed (generally for backbones) the amount of DNA can be increased to 3-5g. The volume of DNA necessary can be obtained from the concentration measured at NANODROP.
2. Mix all the components stated by this restriction tool in a 1.5mL Eppendorf tube and incubate at 37ºC for 1-2h.

Ligation

1. Make your own ligation protocol.

Ligation Protocol

Vector length (bp) :

Vector mass (ng) :

Vector concentration (ng/µL) :

Insert length (bp) :

Insert concentration (ng/µL) :

Ratio Insert/Vector :


Calculate
2. Mix all the components in a tube (always add the T4 ligase at the end).
3. Incubate for at least 3 hours at 16ºC.

Transformation (Top10 chemically competent cells)

1. Get as many aliquots of competent cells (100L) from the -80ºC freezer as transformations to be done and put them on ice for 10-15min.
   NOTE: Don’t forget positive (RFP in the backbone you are using, check plasmids box in the -20ºC freezer) and negative controls (no DNA).
2. Add to the 100µL of competent cells:
   1. 1µL of plasmid or 2µL of ligation product.
   2. 20µL 5X KCM buffer.
   3. Sterile water up to 200µL.
3. Mix by flicking the tube.
4. Incubate on ice for 20-30 minutes.
5. Heat shock at 42ºC for exactly 90 seconds.
6. Return the cells to ice for 1-2 minutes.
7. Add 400µL of fresh LB.
8. Incubate at 37ºC with shaking for 1 hour.
   NOTE: Not at the thermoblock, use a shaking incubator.
9. To increase the concentration of cells before plating, centrifuge the tubes at 4000rpm for 3 minutes, remove 500L of medium, resuspend the pellet in the remaining volume.
10. Plate the rest of the volume of the transformation on selective LB plates and incubate at 37ºC overnight.

Transformation (BL21 competent cells)

1. Put a tube of BL21 Competent E. coli cells on ice for 10 minutes.
2. Add 2µL of plasmid DNA to the cell mixture.
3. Mix by flicking the tube.
4. Place the tube on ice for 30 minutes.
5. Heat shock at 42ºC for exactly 10 seconds.
6. Place on ice for 5 minutes.
7. Add 950µL of SOC medium at room temperature.
8. Incubate at 37ºC with shaking for 1 hour.
   NOTE: Not at the thermoblock, use a shacking incubator.
9. To increase the concentration of cells before plating, centrifuge the tubes at 4000rpm for 3 minutes, remove 700µL of medium, resuspend the pellet in the remaining volume.
10. Plate 70µL of the transformation on selective LB plates and incubate at 37ºC overnight.

Transformation (OneShot^® Top10 Chemically competent cells)

1. Get half the amount of tubes of OneShot^® TOP10 chemically competent cells (tubes with a purple lid) than the transformations to be done from the -80ºC freezer and put them on ice for 10-15min.
2. Divide the volume of competent cells of each tube in 2. Thus, transfer 25L to a new sterile tube.
3. Add 1µL of plasmid or 2µL of ligation product into each tube and mix by flicking.
4. Incubate on ice for 30 min.
5. Heat shock the cells for 30 seconds at 42ºC.
6. Put back on ice for 2 minutes.
7. Add 250µL of LB medium.
8. Incubate at 37ºC with shaking for 1 hour.
   NOTE: Not at the thermoblock, use a shacking incubator.
9. To increase the concentration of cells before plating, centrifuge the tubes at 4000rpm for 3 minutes, remove 200µL of medium, resuspend the pellet in the remaining volume.
10. Plate the rest of the volume of the transformation on selective LB plates and incubate at 37ºC overnight.

Colony PCR (Q5^® High-Fidelity 2X Master Mix)

1. Choose the colonies to be picked for testing and number them. When available pick at least 8 colonies per plate, or more if there are slots left in the PCR machine (it fits 25 samples).
   NOTE: It is more convenient if the numbering is continued between plates of the same batch instead of starting at 1 in each plate. This way there is only one set of numbers per date.
   NOTE: it is possible that not all samples fit in one PCR and then more than one would need to be performed, so keep that in mind for the timing of experiments. But don’t be sad, that would mean that the transformation worked so well that you have a lot of colonies to choose from!! ☺
2. Prepare the mix for all the samples in a single 1.5mL tube (mind pipetting error!). For one sample:

   Component	Volume (µL)
   Q5® HF 2X Master Mix	12.5
   Primer VF2	1.25
   Primer VR	1.25
   Nuclease-free H2O	10
   Total	25

3. Pipette 25µL of mix into each PCR tube.
4. Under sterile conditions, pick a colony making sure that part of it stays on the plate for later use and soak it in the content of the PCR tube. When finished, put them back in the incubator so they grow for a bit longer while the PCR runs.
5. Put the tubes in the PCR machine and apply the following program (applicable for the primers mentioned above, for other sets of primers the annealing temperature needs to be adjusted):

   Step	Temperature (ºC)	Time
   Cell lysis and initial denaturation	95	15min
   Denaturation	95	30sec
   Annealing	53	30sec	x30 cycles
   Extension	72	30sec/1kb
   Final extension	72	5min
   	4	Hold

6. While the PCR is running, prepare a 0.5mL tube with 2µL loading buffer for each PCR sample and one tube with 4µL of 1Kb DNA ladder (or 8µL if 2 gels are needed). This way you can take these tubes into the EtBr area and don’t worry about how to handle the PCR tubes.
7. Cast an agarose gel following the DNA electrophoresis protocol.
8. Prepare an agarose gel with SNAPGENE for comparison with the actual results.
9. Once the PCR program is finished, transfer 8L of PCR product into the tubes containing the loading buffer and follow the DNA electrophoresis protocol.

DNA electrophoresis

Work clean! Handle with gloves all material labelled as EtBr contaminated, don’t take it outside of the EtBr area and don’t touch with gloves anything that is not labelled as EtBr contaminated.

1. Prepare TAE buffer: take the 10X concentrated TAE from the chemicals cabinet and dilute it 10 times with demi water. For 500mL, add 50mL to 450mL of dH2O.
2. Weigh agarose for a 1% gel. For 200mL, 2g of agarose is necessary.
3. Mix the TAE solution with the agarose and heat the solution until it is completely dissolved.
4. Add Ethidium bromide to the gel mould. 2.5L for the small gel and 5L for the big one.
5. Pour the solution into the mould making sure there are no bubbles and that the EtBr is completely mixed. Let it solidify (approx. 20 minutes).
6. Transfer the gel to the electrophoresis cell minding the arrow that indicates the direction of DNA migration, remove the combs and cover it with TAE.
7. Prepare tubes with 5/6 of PCR product and 1/6 of loading buffer.
8. Load the molecular weight marker (ladder) in the first well (check the appropriate volume for each marker, generally 5L works fine) and load the samples in the other wells, according to the order in your lab journal.
   NOTE: Do not contaminate the loading buffer and ladder with EtBr! Do not touch it while wearing a glove.
9. Connect the cables following the colour code and run at 100-130V for 30-60 min.

Restriction product purification

1. For restriction products containing more than one large fragment follow the first 3 steps of the Gel isolation protocol.
   When restriction yields only one large fragment, add an equal volume of Membrane Binding Solution to the volume of restriction product.
2. Pre-warm an aliquot of Nuclease-free water at 50-65ºC.
3. Insert SV Minicolumn into Collection Tube and label both of them according to the labelling of your samples.
4. Transfer the dissolved gel mixture to the Minicolumn assembly. Incubate at room temperature for 1 minute.
   NOTE: When pipetting into the column, aim the pipette to the wall not the membrane to avoid damaging it.
5. Centrifuge the SV Minicolumn assembly at maximum speed for 1 minute.
6. Discard the flowthrough and reinsert the SV Minicolumn into the Collection Tube.
7. Add 700µL of Membrane Wash Solution (if it is the first use, dilute it with 95% ethanol following the bottle’s instructions).
8. Centrifuge the SV Minicolumn assembly at maximum speed for 1 minute.
9. Discard the flowthrough and reinsert the SV Minicolumn into the Collection Tube.
10. Repeat steps 9-11 with 500µL of Membrane Wash Solution and centrifuging for 5 minutes.
11. Once the Collection Tube is empty, centrifuge the Minicolumn assembly at maximum speed for 1 minute with the microcentrifuge lid open to allow ethanol full evaporation.
12. Transfer the SV Minicolumn to an empty 1.5mL tube carefully labelled.
13. Add 500µL of the pre-warmed water (300µL for higher concentrations or when small amounts of DNA are suspected) directly to the centre of the SV Minicolumn, without touching the membrane with the pipette tip.
14. Incubate at room temperature for 5 minutes.
15. Centrifuge at maximum speed for 1 minute.
16. Discard the SV Minicolumn, cap the tube containing the eluted DNA and keep at 4ºC (for immediate use) or -20ºC (for storage).

Cell frozen stock

1. Centrifuge the 50mL tubes at 2000rpm for 10 min.
2. Decant the supernatant without disturbing the pellet.
3. Add fresh LB medium, around 1/3 of the starting volume of the culture.
4. Mix by vortexing until the pellet is completely resuspended.
5. Add sterile 80% glycerol solution, the same volume as fresh LB in step 4.
6. Mix by vortexing.
7. Make 1.5mL aliquots in cryotubes and label them with the cell type, the BioBrick and backbone they contain (if applicable) and the date.
8. Store them at -80ºC and update the inventory in the drive folder.

Plasmid isolation

1. Pre-warm an aliquot of Nuclease-free water at 50-60ºC.
2. Add 1.5 mL of bacterial culture in LB medium to a 1.5 mL micro-centrifuge tube. Centrifuge that tube at max speed for 3 min.
   NOTE: For higher concentrations repeat step 1 in the same tube.
   NOTE: If cryostocks need to be prepared from the same overnight culture tube, remove the necessary volume for plasmid isolation under sterile conditions!
3. Remove the supernatant.
4. Resuspend the pellet in 600 µL of MiliQ water.
5. Add 100 µL of Cell Lysis Buffer, and mix by inverting 6 times. The color change to blue indicates complete lysis.
6. Add 350 µL of cold (4-8ºC) Neutralization Buffer, and mix by inverting the tube. The color change to yellow indicates total neutralization.
7. Introduce a PureYield Minicolumn into a PureYield Collection tube.
8. Centrifuge at maximum speed for 3 minutes, and transfer the supernatant to the Minicolumn assembly.
   NOTE: When pipetting into the column, aim the pipette to the wall not the membrane to avoid damaging it.
9. Centrifuge at maximum speed for 15 seconds and discard the flowthrough.
10. Add 200 µL of Endotoxin Removal Wash to the minicolumn. Centrifuge at maximum speed for 15 seconds. It is not necessary to empty the Collection Tube now.
11. Add 400 µL of Column Wash Solution to the minicolumn, and centrifuge at maximum speed for 30 seconds.
12. Transfer the PureYield Minicolumn to a clean 1.5 mL tube, add 30 µL (or 50L if you expect high concentrations) of pre-warmed MiliQ water directly to the minicolumn matrix. Let stand for 5 minutes at room temperature.
13. Centrifuge at maximum speed in a microcentrifuge for 15 seconds to elute plasmidic DNA. Cap the tube, and store the DNA solution at -20 ºC (or keep it at 4ºC if it is to be used immediately).
    NOTE: If possible, put the lids in line with the circle of the centrifuge (so not pointing out or inwards) so that the caps don’t break while centrifuging.

Overnight cell cultures

1. Prepare 20mL of selective LB (with the antibiotic your cells are resistant to) per colony to be picked.
   NOTE: You can use a sterile bottle to prepare the total volume.
2. Transfer 10mL of selective LB to a 50mL Falcon tube. Prepare 2 tubes per colony: 1 for miniprep and 1 for cryostocks.
3. Pick 1 colony using an inoculation loop and soak it in the LB. Make sure you have cells left for the second tube.
4. Take the tubes to room F1.690 and incubate overnight at 37ºC in the shaking incubator.