Team:Vilnius-Lithuania/Protocols

Protocols

Agarose Gel preparation (x%)

To prepare agarose gels we used TopVision Agarose Tablets that are manufactured by Thermo Fisher Scientific.
Add appropriate number of agarose tablets to the electrophoresis buffer based on the table below to prepare your desired gel percentage

Note: Use a flask that is 2 to 4 times the volume of the solution being prepared.

Before heating soak tablets in a buffer (~ 4 minutes) until tablets completely break into fine-particle slurry. Swirl the slurry to break up any remaining particles. Important: Ensure tablets break up entirely. Heating will render non-dispersed agarose particles insoluble.

Note: Heating times are dependent on the volume of liquid and number of gel tablets to dissolve.

Remove the flask from microwave, swirl gently to dissolve any remaining agarose particles.
Reheat on high power for 1-2 minutes or until the solution is clear and all particles are dissolved.
Remove the flask from the microwave oven, and gently swirl.
Cool the solution to approximately 50-60 °C.
Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). Mix well.
Pour the gel into a tray of required size and place in the well comb, let the gel cool and solidify for 10-15 mins at room temperature.
This gel can now be used to run electrophoresis gels.

Note: To let the gel cool down to the required temperature of about 60 °C one can use a smaller volume container, which is not affected by heat to let a smaller volume of the melted agarose cooldown faster.

PCR

This protocol has been taken from Thermo Scientific and is as follows:
Pipette these items in order listed

*Optionally 5X Phusion GC Buffer can be used. See section 4.2 for details.
**The recommendation for final primer concentration is 0.5 μM, but I can be varied in a range of 0.2-1.0 μM, if needed.
*** Addition of DMSO is recommended for GC-rich amplicons. DMSO is not recommended for amplicons with very low GC % or amplicons that are > 20 kb.

Cycling instructions:

Note: The initial denaturation temperatures as well as annealing temperatures, and extension times are both primer and polymerase dependent, therefore, must be looked up before planning the cycles.

Annealing oligos for lengthier primers

This protocol was taken from Addgene

Place the mixed oligos in a 1.5mL microfuge tube.
Place tube in 90-95°C hot block and leave for 3-5 minutes.
Then, gradually cool to 25°C over 45 minutes.

Colony PCR

Prepare a PCR Master Mix as mentioned in the previous entry noted PCR.

Note: For the possibility of some of the Master Mix volume being lost when dividing the huge stock into PCR tubes make sure to add 10% extra of all the components to the Master Mix as compensation.

Make sure to use final primer concentrations of about 0.4 μM and run 35 cycles of the PCR.
Transfer the Master Mix in 20 μL or 50 μL quantities to small PCR tubes.
Take the dish with bacterial colonies intended for testing.
Transfer a colony that has not yet merged with any other colonies to another plate where numbered and separated regions are marked with marker and afterwards use the same instrument used for transfer and shake the tip that touched the bacteria inside the aliquot of the Master Mix that you have previously pipetted into the PCR tubes.
Repeat for the number of desired colonies.
Place the second dish in the optimal temperature of the bacteria and incubate.
Place the PCR tubes in the PCR thermocycler and run the program adequate to the polymerase used.

Note: If the results have proven to be successful the already transferred colonies can be used for further experiments as they bacteria have usually grown enough for transfers into liquid media for overnight growth.

Gel electrophoresis

Removed the comb from the already cast gel.
Place the gel into the electrophoresis apparatus and make sure that the volume of buffer is sufficient for the electrophoresis, if not add the required amount (TBE or TAE).
Add the DNA size marker or ladder (use about 3-5 μL of the ladder).
After the gel’s wells have been submerged begin loading the samples.
Load 8-50 μL of the sample depending on the purpose of the gel.
Adjust the voltage to 120V.
Set the required time for running the gel.

Note: 20 minutes are sufficient for a gel with already incorporated Ethidium Bromide.

Place the electrodes with the cover over the gel apparatus and initiate the electrophoresis.
After the time has passed turn off the machine.
Take out the gel and place it over a UV or Blue-light illuminator to visualize the EtBr intercalated DNA either for confirmation of experimental results or gel excision.

Gel extraction

Take the gel out of the electrophoresis machine.
To visualize the DNA prior to extraction one cannot use UV illuminators due to the possibility of mutation caused by UV irradiation, a compromise is a Blue-light illuminator in a dark room, which is sufficient enough for accurate excision of the gel.

Note: If the possibility of avoiding UV irradiation is out of the question, please try to keep the exposure of the gel to UV under 10 seconds to avoid damaged DNA.

Excise the bands with the DNA of interest keeping the excess of gel to a minimum as it decreases the yield of DNA after cleanup.
Afterwards follow the Thermo Scientific GeneJET Gel Extraction and DNA Cleanup Micro Kit, which is listed below as follows:
Excise up to 200 mg gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a 1.5 mL tube.
Add 200 μL of Extraction Buffer. Mix thoroughly by pipetting.
Incubate the gel mixture at 50-58°C for 10 minutes or until the gel slice is
Add 200 μL of ethanol (96-100%) and mix by pipetting.
Transfer the mixture to the DNA Purification Micro Column preassembled with a collection tube. Centrifuge the column for 30-60 seconds at 14,000 × g. Discard the flow-through. Place the DNA Purification Micro Column back into the collection tube.

Note:
1. If DNA fragment is ≥ 10 kb centrifuge the column for 2 minutes at 14,000 × g.
2. Close the bag with DNA Purification Micro Columns tightly after each use!

Add 200 μL of Prewash Buffer (supplemented with ethanol, see p. 3) to the DNA Purification Micro Column and centrifuge for 30-60 seconds at 14,000 × g. Discard the flow-through and place the purification column back into the collection tube.

Note: If DNA fragment is ≥ 10 kb centrifuge the column for 1-2 minutes at 14,000 × g.

Add 700 μL of Wash Buffer (supplemented with ethanol, see p. 3) to the DNA Purification Micro Column and centrifuge for 30-60 seconds at 14,000 × g. Discard the flow-through and place the purification column back into the collection tube.

Note: If DNA fragment is ≥ 10 kb centrifuge the column for 1-2 minutes at 14,000 × g.

Repeat step 7.
Centrifuge the empty DNA Purification Micro Column for an additional

Note. This step is essential to avoid residual ethanol in the purified DNA solution. The presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions.

Transfer the DNA Purification Micro Column into a clean 1.5 mL microcentrifuge tube (not included).
Add 10 μL of Elution Buffer to the DNA Purification Micro Column. Centrifuge for 1 minute at 14,000 × g to elute DNA.

Note.

If DNA fragment is ≥ 10 kb the elution volume should be increased between 15-20 μL to get optimal DNA yield. Elution volume less than 10 μL is not recommended.
Lower volume of Elution Buffer for DNA Micro Kit can be used (6-10 μL) in order to concentrate eluted DNA. Please notice that <10 μL elution volume slightly decreases DNA yield.

Discard the purification column and store the purified DNA at -20°C.

Enzymatic reaction cleanup

This protocol was taken from Thermo Scientific and it is as follows:

Adjust the volume of the reaction mixture to 200 μL with Water, nuclease-free or TE buffer (not included).
Add 100 μL of Binding Buffer. Mix thoroughly by pipetting.
Add 300 μL of ethanol (96-100%) and mix by pipetting.
Transfer the mixture to the DNA Purification Micro Column preassembled with a collection tube. Centrifuge the column for 30-60 seconds at 14,000 × g. Discard the flow-through. Place the DNA Purification Micro Column back into the collection tube.

Note.
1. If DNA fragment is ≥ 10 kb centrifuge the column for 2 minutes at 14,000 × g. 2. Close the bag with DNA Purification Micro Columns tightly after each use!

Add 700 μL of Wash Buffer (supplemented with ethanol, see p. 3) to the DNA Purification Micro Column and centrifuge for 30-60 seconds at 14,000 × g. Discard the flow-through and place the purification column back into the collection tube. Note. If DNA fragment is ≥ 10 kb centrifuge the column for 2 minutes at 14,000 × g.
Repeat step 5.
Centrifuge the empty DNA Purification Micro Column for an additional 1 minute at 14,000 × g to completely remove residual Wash Buffer.

Note. This step is essential to avoid residual ethanol in the purified DNA solution. The presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions.

Transfer the DNA Purification Micro Column into a clean 1.5 mL microcentrifuge tube (not included).
Add 10 μL of Elution Buffer to the center of the DNA Purification Micro Column membrane. Centrifuge for 1 minute at 14,000 × g to elute DNA.

Note. *If DNA fragment is ≥ 10 kb the elution volume should be increased to 15-20 μL to get optimal DNA yield. *Lower volume of Elution Buffer for DNA Micro Kit can be used (6-10 μL) in order to concentrate eluted DNA. Please notice that < 10 μL elution volume slightly decreases DNA yield. *Double the elution volume or perform two elution cycles when purifying larger amounts of DNA (for example > 5 μg).

Discard the purification column and store the purified DNA at -20°C.

DNA digestion

This protocol is taken from Thermo Fisher Scientific and is as follows:

Prepare the reaction mixture at room temperature in the order indicated.

Mix gently and spin down.

Incubate at 37°C in a heat block or water thermostat for 5 min. *

Inactive the enzyme (optional). *

Note:
The volume of water should be corrected to keep the indicated total reaction volume. The volume of DNA can be scaled up to 10 μl or down to 0.5 μl depending on the DNA concentration.
Only 2 μL of 10X FastDigest® buffer is required for unpurified PCR product in a 30 μL reaction volume.
See the Certificate of Analysis for enzyme and substrate specific incubation time and enzyme inactivation conditions.

Ligation

This protocol is taken from Thermo Scientific and is as follows:

Thoroughly mix the 5X Rapid Ligation buffer prior to use.
Add the following to a microcentrifuge tube:
Vortex and spin briefly to collect drops
Incubate the mixture at 22°C for 5 min.
Use 2-5 μl of the ligation mixture for transformation.

Note:
*The reaction mixture can be stored at 0-4°C until used for transformation. Prior to electroporation, chloroform extract the ligation mixture and use 1 μl for the electroporation reaction.
*For the incubation of the mixture, it is recommended to keep the ligation mixture in room temperature for about 1 hour to ensure successful and high-yielding ligation results.

Chemical transformation

Take out bacteria out of the -80°C freezer and immediately place under ice to let them fully thaw.
Turn on the UV sterilization function in the working box for about 15 minutes to sterilize the working environment before opening the test tube with the bacteria to avoid any risk of contamination.
Take out petri dishes with required antibiotic out of the refrigerator.
Mix 50 μl with 10 μl of the ligation reaction mixture in the sterile box and then further incubate on ice for 5-10 minutes.
Prepare as many of transformation controls as possible: transform the bacteria without DNA (contamination control), only the linearized digested vector (to check if the DNA is properly digested), positive control with purified non-digested DNA (to check whether everything is all right with competent cells and the transformation).
Heat up 2 thermoblocks with 42°C and 37°C.
Perform a heat shock for 1 min on 42°C.
Incubate on ice for 5-10 minutes.
Suspend the bacteria with 500-1000 μl of LB (Luria Broth).
Incubate in the 37°C heat block for 20-30 minutes.
Centrifuge the cells for 5 minutes at 3000rpm.
Discard the supernatant in one quick movement, there should be some of the LB medium still left, resuspend the bacteria in the remaining LB medium and plate out on a LB-petri plate with the correct antibiotic.
Incubate the plates overnight at the optimal temperature for selected bacteria.

LIC cloning

This protocol was taken form Thermo Scientific and is as follows:

To generate the necessary 5' and 3' overhangs on the purified PCR template, prepare the following reaction mixture at room temperature:

Vortex briefly and centrifuge for 3-5s.
Incubate the reaction mixture at room temperature (20-25°C) for 5 min.

Note. Do not exceed 5 min.

Stop the reaction by adding 0.6 μL of 0.5M EDTA, mix well.
Note. Store the prepared PCR product in the reaction mixture at -20°C if the annealing step with the LIC vector cannot be performed immediately. Thaw and mix carefully prior to performing the annealing reaction.
Set up the annealing reaction:
Add 1 μL pLATE, LIC-ready vector (60 ng, 0.02 pmol DNA) to the T4 DNA polymerase treated PCR product prepared in steps 1–3 of this protocol. Vortex briefly and centrifuge for 3-5 s.
Incubate the annealing mixture at room temperature (20-25°C) for 5 min.

Note. Annealing is complete within 5 min of incubation. Reactions can be incubated up to 2 hours without affecting results. Longer incubation times do not improve efficiency.

Use the annealed mixture directly for bacterial cell transformation.

Protein Electrophoresis (SDS Page)

Separating gel

Prepare the gel casts in holders. Fill the holder with water to check for leakage after 5 min. After leakage check, pour all water from gel casts and dry residual with paper towel.

To prepare separation-gel, mix the reagents listed in the table below with the proper volumes to receive a gel with desired composition.

NOTE! Add TEMED and APS last, solidification of the gel will occur when these reagents are added.

Pipette gel mixture into gel casts up to a height approximately 1 cm below the gels comb.

Fill the remaining cast with isopropanol (if not available use distilled water).

Allow to harden for 30-60 min.

Pour all isopropanol (or water) from gel casts and dry residual with paper towel.

Ammonium Persulfate (APS) - 20 mg/mL
Acrylamide 30 % solution
TEMED
Separating Gel Buffer - 1.5 M Tris-HCl pH 8.8 with 0.4% SDS. Stored at 4°C.
*Stacking Gel Buffer - 0.5 M Tris-HCl pH 6.8 with 0.4% SDS. Stored at 4°C

Stacking gel

To prepare stacking-gel, in a tube mix the reagents listed in table 4 below with the proper volumes to receive a gel with desired composition.

NOTE! Add TEMED and APS last, solidification of the gel will occur when these reagents are added.

Pipette gel mixture on top of separating gel to fill cast and remove all air bubbles.

Fill the remaining cast with isopropanol (if not available use distilled water).

Insert the comb whilst the gel is still in liquid form. The comb will form wells to load samples when the gel turns solid.

Allow to harden for 45-60 min.

Electrophoresis

Running the gel

Mount the gels into the tank, remove combs and fill the inner chamber of the tank with 1x running buffer to the top. Fill a third of the volume in the outer chamber of the tank with 1x running buffer.

Pipette 2 µl ladder and 10 µl sample into each well.

Connect power pack (red-red, black-black) and run at 100 V, 60 min or until the blue line almost reaches the bottom of the gel.

Once electrophoresis has finished, remove gels from the glass plates.

Carefully wash all equipment used for electrophoresis with water.

10x Running buffer

Dissolve 30.0 g of Tris base, 144.0 g of glycine, and 10.0 g of SDS in 1000 mL of H2O. The pH of the buffer should be 8.3 and no pH adjustment is required. Store the running buffer at room temperature and dilute to 1X before use.

Coomassie Blue Staining

Prepare the staining solution containing 0.1 % Coomassie Blue in 40 % ethanol, 10 % acetic acid.

After electrophoresis, incubate 1 or 2 gels in a staining container containing 100 mL Coomassie Blue staining
solution.
Incubate at room temperature for 1 hour until bands are visible.

Decant the stain and rinse the gel once with deionized water.

Prepare 100 mL destaining solution containing 10 % ethanol and 7.5 % acetic acid.

Gently shake the gel at room temperature on an orbital shaker until the desired background is achieved.

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