PROTOCOLS

In this page you will find the protocols that worked well for us.

Agarose Gel Preparation

Materials

• TAE 1X
• Agarose
• GelRed

Procedure

1. For a 1.2 % agarose gel (small fragments), put 0.6 grams of agarose into 50 mL of TAE 1X. For a 3 % agarose gel (big fragments), put 1.5 g of agarose into 50 mL of TAE 1X.
2. Warm the solution in the microwave (max. Power) for 30 seconds, or until agarose has melted.
3. Mix the solution.
4. Add 1.3uL of Gel Red (1.5 for 3 % gel).
5. Pour the solution into an agarose gel mold including the comb.
6. Wait 20 min for the gel to solidify.
7. Place the gel in TAE 1X and remove comb.
8. Mix ~200ng of DNA with nuclease free water and loading buffer to a final 1X concentration (total volume of 12uL).
9. Run the gel for 45 min at 100 Volts (more time if needed). For a 3 % gel, run for 2 hours at 80 Volts.
10. Take a picture of the gel at the UV detector.

Gibson Assembly Protocol

Materials

• Plasmids of interest (miniprep)
• NEB Gibson assembly Kit (Gibson Assembly Master Mix (2X) and chemically competent cells)
• Deionized H2O

Procedure

1. Set up the following reaction on ice:

	2-3 Fragments assembly* (X uL)	4-6 Fragments assembly* (X uL)
Total Amount of Fragments	0.02-0.5 pmols	0.2-1 pmols*
Gibson Assembly Master Mix (2X)	10 uL	10 uL
Deionized H2O	10-X uL	10-X uL
Total Volume	20 uL	20 uL

*Optimized cloning efficiency is 50-100ng of vectors with 2-3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps.

2. Incubate samples in a thermocycler at 50C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at -20°C for subsequent transformation.
3. Transform 50 uL of competent cells (provided with the kit) with 2 μl of the assembly reaction, following the transformation protocol.

You can find the original NEB Gison Assembly Protocol here

Miniprep – with QlAprep Spin Miniprep kit

Materials

• Ovenight liquid cell cultures (5-8 mL)
• QlAprep Spin Miniprep kit

Procedure

1. Pellet bacterial cells from overnight 5-8 mL culture (4000 rpm, 10 min) and discard the medium.
2. Resuspend pelleted bacterial cells in 250 μL Buffer P1 (stored at 4°C). Ensure that RNase A has been added to Buffer P1. No cells clumps should be visible after resuspension of the pellet. Transfer the resuspended cells into a 1.5 microcentrifuge tube.
3. Add 250 μL Buffer P2 and gently invert the tube 4-6 times to mix (do not vortex). If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Let stand for 2-4 minutes; never more than 5.
4. Add 350 μL Buffer N3 and invert the tube immediately but gently 4-6 times. The solution should become cloudy.
5. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge. A compact pellet will form.
6. Apply the supernatant from step 5 to the QlAprep Spin Column by pipetting.
7. Centrifuge for 1 min at 13,000 rpm. Discard the flow-through.
8. Wash QlAprep Spin Column by adding 750 μL Buffer PE and centrifuge for 1 min at 13,000 rpm.
9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
10. Place the QlAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μL Buffer EP (10 mM Tris-Cl, pH 8.5) to the center of each QlAprep Spin Column, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm.

You can find the original Qiagen Miniprep Protocol here.

PCR Protocol

Phusion PCR

Materials

• Phusion® High-Fidelity DNA Polymerase PCR Kit
• Nuclease-free Water
• Primers
• Template DNA

Procedure

1. Setup the following reaction in a 0.5 mL PCR tube:

   Component	20 µL Reaction	50 µL Reaction	Final Concentration
   5X Phusion HF or GC Buffer	4 µL	10 µL	1X
   10 mM dNTPs	0.4 µL	1 µL	200 µM
   10 µM Forward Primer	1 µL	2.5 µL	0.5 µM
   10 µM Reverse Primer	1 µL	2.5 µL	0.5 µM
   Template DNA	variable	variable	<250 ng
   DMSO (optional)	(0.6 µL)	(1.5 µL)	3 %
   Phusion DNA Polymerase	0.2 µL	0.5 µL	1.0 units/50 µl PCR
   Nuclease-free Water	To 20 µL	To 50 µL

2. Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.
3. Transfer PCR tubes from ice to a PCR machine with the block preheated to 98°C and begin thermocycling:

   STEP	TEMP	TIME
   Initial Denaturation	98 °C	30 seconds
   25-35 Cycles	98°C	5-10 seconds
   	45-72 °C Annealing Temp	20 seconds
   	72 °C Elongation Temp	15-30 seconds per kb
   Final Extension	72 °C	5-10 minutes
   Hold	4-10 °C

You can find the original PCR Protocol for Phusion® High-Fidelity DNA Polymerase (M0530) here.

Taq PCR

Materials

• ThermoPol Buffer 10X
• dNTPs
• Taq Polymerase
• Template DNA
• Primers
• Nuclease-free water

Procedure

1. Prepare the following reaction in a 0.5 mL PCR tube on ice:

   Component	25 µL Reaction	50 µL Reaction	Final Concentration
   10X ThermoPol or Standard Taq Reaction Buffer	2.5 µL	5 μL	1X
   10 mM dNTPs	0.5 µL	1 µL	200 µM
   10 µM Forward Primer	0.5 µL	1 µL	0.2 µM
   10 µM Reverse Primer	0.5 µL	1 µL	0.2 µM
   Template DNA	variable	variable	<1000 ng
   Taq DNA Polymerase	0.125 µL	0.25 µL	1.25 units/50 µL PCR
   Nuclease-free Water	To 25 µL	To 50 µL

   * Due to the difficulties in pipetting small volumes of enzyme, Taq DNA Polymerase can be diluted in 1X reaction buffer. For example, 1 µl of Taq DNA Polymerase is mixed with 4 µl of 1X reaction buffer and 1 µl of that mixture is added to the reaction. Enzyme diluted 1X reaction buffer should not be stored for future use.
2. Gently mix the reaction and spin down in microcentrifuge. If the thermocycler does not have a heated cover, add one drop of mineral oil to the reaction tube to prevent evaporation.
3. Cycling Conditions for a Routine PCR:

   STEP	TEMP	TIME	CYCLES
   Initial Denaturation	95 °C	30 seconds	1
   Denaturation	95 °C	15-30 seconds	30
   Annealing	45-68 °C	15-60 seconds
   Extension	68 °C	1 minute per kb
   Final Extension	72 °C	5 minutes
   Hold	4-10°C

You can find the original NEB Taq PCR PCR Product Purification Protocol

Materials

• PCR products
• Qiagen QlAquick PCR kit

Procedure

1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
2. To bind DNA, apply the sample to the QlAquick column and centrifuge for 1 min at 13,000 rpm.
3. Discard the flow-through. Place the QlAquick column back into the same tube.
4. To wash, add 750 uL Buffer PE to the QlAquick column and centrifuge for 1 min at 13,000 rpm.
5. Discard flow-through and place the QlAquick column back in the same tube. Centrifuge the column for an additional 1 min.
6. Place QlAquick column in a clean 1.5 mL microcentrifuge tube.
7. To elute DNA, add 30 uL Buffer EP (10 mM Tris-Cl, pH 8.5) to the center of the QlAquick membrane, let the column stand for 1 min and then centrifuge for 1 min at 13,000 rpm.

You can find the original Qiagen PCR Product Purification Protocol here.

Thermocompetent E coli cells preparation

Materials

• Bacterial colony from plate (day 1)
• LB medium (day 1)
• DMSO (day 2)
• Liquid nitrogen (day 2)
• Sterile Inoue buffer (day 2) – for buffer preparation, see below

Note: the 250 mL of culture can be separated after growth into smaller tubes (for example, to facilitate centrifugation at 4 ºC). Make sure to respect the proportion of all added buffers and solutions.

Procedure

1. Pick a single colony from a plate that has been incubated overnight at 37 ºC.
2. Transfer the colony into 30 mL of LB in a 250 mL flask.
3. Use this starter culture to inoculate three 1-liter flasks with 1 mL of starter culture, each containing 250 mL of LB (there’s no need to make triplicates, one flask is enough to get 100-120 x 200 µL tubes).
4. Incubate flasks overnight (depends on growth, 8-17 hours) at 22 ºC (room temperature) with shaking at 180 rpm.
5. Monitor the OD600 (every 45 min) until it reaches 0.55, and transfer the culture on ice for 10 min. It is important to stop growth at 0.55, can be longer than 10 min on ice.
6. Centrifuge the cells at 2500 g for 10 min at 4 ºC (temperature is important, it’s better if you can do the rest of the protocol in a cold room).
7. Pour off the medium and remove any remaining drop.
8. Resuspend cells gently in 240 mL of ice-cold Inoue buffer (0 ºC).
9. Repeat steps 6 to 8, using only 60 mL of ice-cold Inoue buffer for the resuspention.
10. Add 4.5 mL of DMSO and store on ice for 10 min.
11. Dispense 200 µL aliquots of the suspension into sterile microtubes.
12. Put the tubes in a container and snap-freeze them by pouring liquid nitrogen on them. It is important to pour the liquid nitrogen on them, as they have to freeze very fast; the aliquots are best when small for the same reason.
13. Store the tubes at -80 ºC until needed. Note: thaw on ice when used.

Inoue Transformation Buffer

Materials

• MnCl2 · 4H2O
• CaCl2 · 2H2O
• KCl
• Milli-Q water (or bidistilled)
• Pre-rinsed .45 µm Nalgene filter - 1 or 2, depending on PIPES solution preparation

Procedure

1. Make .5M PIPES (piperazine‐1,2‐bis[2‐ethanesulfonic acid]) at pH 6.7
1. Dissolve 15.1 g of PIPES in 30 mL Milli-Q water.
2. Adjust pH of solution to 6.7 (with KOH or HCl; you can easily add 30 mL of KOH to begin with).
3. Add Milli-Q water to final volume of 100 mL.
4. Optional: sterilize solution by vacuum filtration in pre-rinsed Nalgene filter of .45 µm pore size. Not necessary if used for Inoue Buffer.
2. Dissolve solutes as follows in 800 mL of Milli-Q water.

Reagent	Qty/L	Final Conc.
MnCl2 · 4H2O	10.88 g	55 mM
CaCl2 · 2H2O	2.20 g	15 mM
KCl	18.65	250 mM

3. Add 20 mL PIPES (.5 M, pH 6.7).
4. Adjust total volume to 1 L with Milli-Q water.
5. Sterilize Inoue Transformation Buffer through pre-rinsed .45 µm Nalgene filter.

Transformation Protocols

NEB transformation protocol

Materials

• DNA
• Competent cells
• SOC medium (SOB + Glucose) - room temperature
• Petri dish with antibiotic resistance - 37 ºC

Procedure

1. Thaw competent cells on ice.
2. Chill 5 ng of ligation mixture in a 1.5 ml microcentrifuge tube and then add 50 µL of competent cells. Do not vortex or mix.
3. Place the mixture on ice for 30 min (do not mix).
4. Heat-shock cells at 42 ºC for 30 s in a pre-heated water bath (works better than on a heating block) (do not mix).
5. Place on ice and add 950 µL of room temperature SOC medium to the tube (make sure that it is not contaminated).
6. Place tube at 37 ºC for 60 min. Shake or rotate (250 rpm). Warm selection plates to 37 ºC (not necessary, but increases efficiency).
7. Spread 50-100 µL of samples on plates (dependent on efficiency of the competent cells); incubate overnight at 37 ºC.

You can find the original NEB Transformation Protocol here.

iGEM Transformation Protocol

Materials

• DNA
• Competent cells
• SOC medium (SOB + Glucose)
• Petri dish with antibiotic resistance

Procedure

1. Thaw competent cells on ice.
2. Add 50 µL of thawed competent cells into a pre-chilled 2 mL tube.
3. Add 5 ng of resuspended DNA to the sample and pipet gently up and down a few times.
4. Keep the sample on ice for 30 min.
5. Heat-shock cells at 42 ºC for 60 s in a pre-heated water bath (works better than on a heating block).
6. Place samples on ice for 5 min (can be more).
7. Add 200 µL of SOC medium to the sample (make sure that it is not contaminated).
8. Incubate cells at 37 ºC for 2 hours with shaking or rotating. 2 hours recovery time after incubation helps in transformation efficiency.
9. Plate 20 µL and 200 µL of the sample on two plates and spread.
10. Incubate the plates at 37 ºC for 12-14 hours (agar side of the plate has to be up). Note: Incubating for too long will increase the occurrence of satellite colonies (especially if resistant to Ampicillin).

You can find the original iGEM Transformation Protocol here.