Experiments | Tec-Monterrey

PCR

We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed prior to use.

Materials

Q5 High-Fidelity 2X Master Mix
10 µM Forward Primer
10 µM Reverse Primer
DNA Template
H2O (Nuclease-free)

Procedure

Add Q5 High-Fidelity 2X Master Mix to a PCR tube.
Add each Primer to a final concentration of 500 nM.
Add up to 10 ng of Template DNA.
Add Nuclease-Free Water up to final reaction volume (2 times the volume of master mix).
Gently mix the reaction.
- Alternatively if doing several reactions and using the same amount of template for each one, a pre-mixture of the remaining reagents can be prepared and then distribute the amount of mix in separate tubes prior to adding the template DNA.
Collect all liquid to the bottom of the tube by a quick spin if necessary

Quickly transfer PCR tubes to a thermocycler previously set to the following configuration:

Step	Temperature	Time
Initial denaturation	98°C	30 Seg
	98°C	10 Seg
25 to 35 Cycles*	60°C	20 Seg
	72°C	25 Seg
Final extension	72°C	2 Minutes
Hold	4°C	∞

*Use less than 20 cycles if amplifying pure synthesized parts

PCR product purification

Input amount of DNA to be purified should not exceed the binding capacity of the column (5 μg). A starting sample volume of 20–100 µL is recommended. For smaller samples, TE can be used to adjust the volume to the recommended volume range. Centrifugation should be carried out at 16,000 x g (~13K RPM in a typical microcentrifuge) in a standard laboratory microcentrifuge at room temperature.

Materials

Monarch® DNA Cleanup Columns (5 μg)
Monarch Collection Tubes (2mL)
Monarch® DNA Wash Buffer
Monarch® DNA Cleanup Binding Buffer
Monarch® DNA Elution Buffer or Nuclease-Free Water
Ethanol (≥ 95%)

Before you begin

Add 20 mL of ethanol to the Monarch DNA Wash Buffer (4 volumes of ≥ 95% ethanol per volume of Monarch DNA Wash Buffer).

Procedure

Dilute sample with DNA Cleanup Binding Buffer according to the table:

Sample type	Binding buffer:Sample ratio	Example
dsDNA > 2 kb (plasmids, gDNA)	2:1	200 µL Buffer: 100 µL sample
dsDNA < 2 kb (some amplicons, fragments)	5:1	500 µL Buffer:100 µL sample
ssDNA > 200 nt	7:1	700 µL Buffer: 100 µL sample

Mix well by pipetting up and down or flicking the tube. Do not vortex. A starting sample volume of 20–100µL is recommended.
Insert column into collection tube and load sample onto column and close the cap. Spin for 1 minute, then discard flow-through.
Re-insert column into collection tube. Add 200µL DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
Repeat previous step.
Transfer column to a clean 1.5 mL microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to the next step.
Add ≥ 6 µL of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.
- Note: Typical elution volumes are 6–20 µL. Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated

Electrophoresis

QUICKSILVER™ TAE Buffer Powder and SmartGlow PS (E4500) are used for this protocol. Protocol will change with other products.

Materials

QUICKSILVER™ TAE Buffer Powder
Distilled water
SmartGlow PS
Agarose
SmartCheck Ladder

Procedure

Add contents of buffer pouch to a graduated cylinder or beaker.
Slowly add distilled water to 1 liter.
Mix well until all granules are dissolved.
Add additional water to bring the volume to 1L, if necessary.

Add 0.5g of agarose to 50 mL of TAE buffer and microwave to dissolve.
Add 2.5 µL of SmartGlow and mix.
Pour gel into gel tray.
Let it sit until solidified and place into the tank.
Add TAE buffer into the tank until the gel is covered.

NOTE: For better results, 5uL of SmartGlow can be added per 100mL of TAE running buffer.

Add 5-10 µL of Ladder
Mix X6 loading dye with samples and DNA markers at a 1:5 (dye to sample) dilution.
Run at 100V for 45 to 60min.
View the gel using a UV or blue light.

DNA from gel purification

All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM). Note that the columns hold up to 800uL.

Materials

Monarch® DNA Cleanup Columns (5 μg)
Monarch Collection Tubes (2mL)
Monarch® DNA Wash Buffer
Monarch® Gel Dissolving Buffer
Monarch® DNA Elution Buffer or Nuclease-Free Water
Ethanol (≥ 95%)

Before You Begin

Add 20 mL of ethanol to the Monarch DNA Wash Buffer (4 volumes of ≥ 95% ethanol per volume of Monarch DNA Wash Buffer).

Procedure

Excise the DNA fragment to be purified from the agarose gel using a razor blade, scalpel or other clean cutting tool. Use care to trim excess agarose. Transfer it to a 1.5 mL microcentrifuge tube and weigh the gel slice.
Add 4 volumes of Monarch Gel Dissolving Buffer to the tube with the gel slice (e.g., 400 µL buffer per 100 mg agarose). If the gel slice is >150 mg, consider reducing the amount of Gel Dissolving Buffer to 3 or 3.5 volumes to minimize the guanidine salt present.
Incubate the sample between 37–55°C (typically 50°C), inverting periodically until the gel slice is completely dissolved (generally 5–10 minutes).
Insert the column into collection tube and load sample onto the column. Spin for 1 minute, then discard flow-through.
Re-insert column into collection tube. Add 200 µL DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
Repeat previous step.
Transfer column to a clean 1.5 mL microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to the next step.
Add ≥ 6 µL of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.
- Note: Typical elution volumes are 6–20 µL. Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated

Digestion

Set up reaction as follows:

Component	20 µL REACTION
DNA	>1 µg
10X rCutSmart Buffer	2 µL (1X)
EcoRI-HF	0.5 µL (10 units)
Nuclease-free Water	to 20 µL

Incubate at 37°C for at least 2 hours (EcoRI maintains its activity for at least 4-8 hours).
- For convenience, 0.5µL is specified; adjust as needed. In general, 5–10 units of enzyme per µg DNA, and 10–20 units for genomic DNA in a 1 hour digest is recommended.
- Reaction volumes can be linearly adjusted up or down, however enzyme volume should not exceed 10% of the total reaction volume to prevent star activity due to excess glycerol.
Optional: Heat inactivate at 65°C for 20 min.
- Heat inactivation is recommended if you plan to continue to another step in the workflow without a DNA purification step.

Double-digestion

Set up reaction as follows:

Component	20 µL REACTION
DNA	>1 µg
10X NEBuffer 2.1	2 µL (1X)
EcoRI-HF	0.5 µL (10 units)
PstI	0.75 µL (15 units)
Nuclease-free Water	to 20 µL

Note: PstI has only 75% of activity in NEBuffer 2.1 and EcoRI has 100%, PstI is added in a ratio of 1.5:1 to EcoRI in order to compensate for the difference.

Incubate at 37°C for at least 2 hours (EcoRI maintains its activity for at least 4-8 hours and PstI for at least 2-4 hours).
- For convenience, 0.5µL is specified; adjust as needed. In general, 5–10 units of enzyme per µg DNA, and 10–20 units for genomic DNA in a 1 hour digest is recommended.
- Reaction volumes can be linearly adjusted up or down, however enzyme volume should not exceed 10% of the total reaction volume to prevent star activity due to excess glycerol.
Optional: Heat inactivate at 80°C for 20 min.
- Heat inactivation is recommended if you plan to continue to another step in the workflow without a DNA purification step.

Competent Cells Preparation

Materials

SOB
- 0.5% (w/v) yeast extract
- 2% (w/v) tryptone
- 10 mM NaCl
- 2.5 mM KCl
- 20 mM MgSO4
CCMB80 buffer
- 10 mM KOAc pH 7.0
- 80 mM CaCl2
- 20 mM MnCl2
- 10 mM MgCl2
- 10% glycerol
- Adjust pH DOWN to 6.4 with 0.1N HCl if necessary
  - Slight dark precipitate does not affect its function, but makes the next step more difficult.
- Sterile filter and store at 4°C

Procedure

Inoculate 250 mL of SOB medium with 1 mL vial of seed stock and grow at 20°C to an OD600nm of 0.3.
- This takes approximately 16 hours.
- Room temperature will work. You can adjust this temperature somewhat to fit your schedule.
- Aim for lower, not higher OD if you can't hit this mark.
Pre-chill in ice as many centrifuge tubes as needed.
Transfer the culture to the chilled centrifuge tubes.
Centrifuge at 3000g at 4°C for 10 minutes.
Discard supernatant.
Add 40mL of ice cold CCMB80 buffer first to resuspend the cells.
When cells are in suspension, add another 40mL of CCMB80 buffer for a total of 80mL.
Pipet buffer against the wall of the centrifuge bottle to resuspend cells.
- Avoid pipetting directly into cell pellets.
Incubate on ice for 20 minutes.
Centrifuge at 3000G at 4°C for 10 minutes.
Discard supernatant.
Resuspend cell pellet in 10 mL of ice cold CCMB80 buffer.
- If using multiple centrifuge tubes, combine the cells post-resuspension.
Incubate on ice for 20 minutes. Prepare for aliquoting
Aliquot 200 µL into chilled PCR tubes.
Store at -80°C indefinitely.

Note:

Transformation

Procedure

Thaw a tube of competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µL of cells into a 1.5 mL microcentrifuge tube on ice.
Add 1-5 µL containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
Place the mixture on ice for 30 minutes. Do not mix.
Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
Place on ice for 5 minutes. Do not mix.
Pipette 950 µL of room temperature SOC into the mixture.
Place at 37°C for 60 minutes shaking at 250 RPM.
Prepare LB Agar selection plates with the adequate antibiotic.
Spread 100 µL of the culture onto a selection plate and incubate overnight at 37°C.
- Dilute prior to plating if necessary.

Miniprep

Isolation and propagation of DNA molecules are essential features of recombinant DNA technologies. To this purpose, DNA molecules are copied (propagated) using the replication machinery of its host. A plasmid or a vector is a circular DNA molecule that contains the information necessary for its replication inside a host. Plasmids and vectors contain antibiotic resistance genes allowing selection of bacteria hosting the plasmid of interest when a population of bacteria is cultured in the presence of the antibiotic. Plasmids and vectors also contain specific DNA sequences recognized by restriction enzymes allowing the insertion of DNA fragments through the combination of a host plasmid cut by a set of restriction enzymes and a DNA fragment with compatible ends in a reaction mediated by a ligase. The newly ligated plasmid containing the DNA insert will be transformed into a host to replicate and propagate itself. This host-assisted heterologous-DNA fragment replication process is known as cloning.

Materials

Solution I
- Tris HCl 25 mM
- EDTA 10 mM
Solution II
- NaOH 0.2 N
- SDS 41.9 mM
  - SDS must be dissolved carefully.
Solution III
- Potassium acetate 3 M
  - Must be previously dissolved in water.
- Acetic Acid 2 M
1.5 mL microcentrifuge tubes
Isopropanol
Ethanol 70%
Nuclease-Free Water
RNAse

Before you begin

Add 10 mL of LB media to a 50mL tube with the corresponding antibiotic, inoculate with a single colony from a plate. All cultures were incubated overnight at 37ºC and shaken at 250 rpm.

Procedure

Centrifuge for 5 min at 5000xg.
Discard supernatant.
Add 200 μL of Solution I and 1 μL of RNAse.
Resuspend until the pellet is completely solubilized.
Incubate 5 min at room temperature.
Add 200 μL of Solution II.
Mix by inversion.
Incubate 5 min at room temperature.
Add 200 μL of Solution III.
IMix by inversion.
Incubate 10 min at -20ºC.
Centrifuge for 10 min at 16000xg (14,000 RPM).
Transfer supernatant to a new 1.5 mL tube.
- Avoid disturbing/touching white precipitate.
Add 500 μL of isopropanol.
Centrifuge for 10 min at 16000xg (14,000 RPM).
Discard supernatant.
Add 1 mL of ethanol 70%.
Mix by vortexing.
Centrifuge for 5 min at 16000xg (14,000 RPM).
Discard supernatant.
Dry tubes with open cap at 37ºC for 45 min.
Add 50 μL of Nuclease-Free water.
Mix by vortexing.
Storage at 4ºC for short periods of time (days) or -20ºC indefinitely.

Colony PCR

Materials

PCR tubes
Nuclease-Free water
Sterile loop

Procedure

Add 10-20 µL of Nuclease-Free water into a PCR tube.
Pick a single colony with a sterile loop.
Inoculate the water with the loop, making sure most bacteria ends in the water.
- The water must become somewhat cloudy.
Incubate at 95°C for 10 minutes.
- This could be easily done in a thermocycler.
Perform a usual PCR procedure using 1-2 µL of the lysed bacteria as template.

IPTG Induction

IPTG or Isopropyl β-D-1-thiogalactopyranoside is a chemical reagent mimicking allolactose, which removes a repressor from the lac operon to induce gene expression, since our promoters work by the interaction of the lac repressor IPTG Induction allow us to express our protein of interest.

Procedure

Transform expression plasmid into E.coli BL21(DE3).
Plate on antibiotic selection plates and incubate overnight at 37°C.
Resuspend a single colony in 10 mL liquid culture with proper antibiotic.
Incubate at 37°C until OD600 reaches 0.4–0.8.
Induce with 10 µL of a 1 M stock of IPTG (final concentration of 1 mM) and induce for 2-4 hours at 37°C.
Analyze by method of choice.

Purification by SPRI beads technology

The magnetic microparticles with the SPRI (solid-phase reversible immobilization) technology are a high-throughput DNA and RNA purification strategy. AMPure XP utilizes an optimized buffer to selectively bind DNA fragments 100 bp and larger to paramagnetic beads. Excess primers, nucleotides, salts, and enzymes can be removed using a simple washing procedure. The result is a more purified and/or concentrated sample.

Materials

AMPure XP beads
Fresh 80% ethanol
Nuclease free water

Procedure

Determine whether or not a plate transfer is necessary.
Shake the AMPure XP bottle to resuspend any magnetic particles that may have settled. Then add AMPure XP according to the sample reaction volume.
Mix reagent and sample thoroughly by pipette mixing 10 times. Let the mixed samples incubate for 5 minutes at room temperature for maximum recovery. The color of the mixture should appear homogeneous after mixing.
Mix reagent and sample thoroughly by pipette mixing 10 times. Let the mixed samples incubate for 5 minutes at room temperature for maximum recovery. The color of the mixture should appear homogeneous after mixing.
Place the reaction plate onto a magnet plate for 2 minutes to separate beads from the solution. Wait for the solution to clear before proceeding to the next step.
With the reaction situated on the magnet, aspirate and discard the cleared solution from the reaction plate and discard.
Dispense 200 μL of 80% ethanol to each well of the reaction plate and incubate for 30 seconds at room temperature. Aspirate out the ethanol and discard.
Repeat the last step for a total of two washes.
Remove the reaction plate from the magnet plate, and then add the necessary volume (10 - 40 µL)of NFW to each well of the reaction plate and pipette mix 10 times. Incubate for 2 minutes.
Place the reaction plate onto a magnet plate for 1 minute to separate beads from the solution.
Transfer the eluate to a new plate.

M13 bacteriophage propagation and preparation

Stocks of bacteriophage M13 are usually grown in liquid culture. The infected bacteria do not lyse but, instead, grow at a slower than normal rate to form a dilute suspension, this propagation is necessary in order to perform infection protocols and phage display procedures.

Procedure

Incubate a 2 mL culture of E. coli TG1 until a log phase is reached, this allows for a more efficient infection.
Add 2 μL of VCSM13 bacteriophage stock and incubate at 37°C and 250 rpm for 1 hour.
Transfer the 2 mL infected culture to 2 falcon tubes each with 15 mL LB media.
Incubate at 37°C and 250 rpm for 1 hour.
Add kanamycin to a final concentration of 35 μg/mL to each falcon tube.
Incubate overnight at 37°C and 250 rpm.
Centrifugate the tubes at 2500g for 15 minutes.
Transfer the supernatants to new falcon tubes and incubate in a water bath at 70°C for 20 minutes.
Centrifugate the tubes at 2500g for 15 minutes and transfer the supernatants to new falcon tubes.
Store at 4°C.

M13 bacteriophage titer determination

Procedure

Inoculate 2 mL of LB medium with 2 μL of E. coli TG1 at 37°C and 250 rpm for 1 hour.
Prepare 10-7, 10-8 and 10-9 dilutions of the bacteriophage preparation in LB medium.
Use 1 μL of the dilutions to infect 50 μL of the TG1 culture and incubate for 15 minutes at room temperature.
Add 3 mL of LB medium and pour into LB agar plates andIncubate overnight at 37°C
Determine the titer from the number of plaques. 50 plaques derived from the 10-8 solution corresponds to a titer of 5x1012 plaque-forming units (pfu) per mL. The titer should be in the range of 1012 to 1013 pfu/mL.
The titer tends to decrease with time and the bacteriophage preparation is stable for many months at 4°C.

M13 bacteriophage purification

Procedure

Incubate the bacteriophage preparation with PEG 6000 at 4°C with continuous agitation for 90 minutes.
Centrifugate at 10000 rpm for 10 minutes and discard the supernatant.
Resuspend the pellet in deionised water.
Lower the pH to 4 using 5M HCl.
Centrifugate at 15000 rpm for 5 minutes and discard the supernatant.
Resuspend the pellet in deionised water and centrifugate again at 15000 rpm for 5 minutes.
Resuspend the pellet in deionised water and store at -20°C until ready to use.