Difference between revisions of "Team:Vilnius-Lithuania/Protocols"

Agarose Gel preparation (x%)

1. To prepare agarose gels we used TopVision Agarose Tablets that are manufactured by Thermo Fisher Scientific.
2. 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.




3. 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.

4. Remove the flask from microwave, swirl gently to dissolve any remaining agarose particles.
5. Reheat on high power for 1-2 minutes or until the solution is clear and all particles are dissolved.
6. Remove the flask from the microwave oven, and gently swirl.
7. Cool the solution to approximately 50-60 °C.
8. 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.
9. 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.
10. 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

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

Colony PCR

1. 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.

2. Make sure to use final primer concentrations of about 0.4 μM and run 35 cycles of the PCR.
3. Transfer the Master Mix in 20 μL or 50 μL quantities to small PCR tubes.
4. Take the dish with bacterial colonies intended for testing.
5. 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.
6. Repeat for the number of desired colonies.
7. Place the second dish in the optimal temperature of the bacteria and incubate.
8. 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

1. Removed the comb from the already cast gel.
2. 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).
3. Add the DNA size marker or ladder (use about 3-5 μL of the ladder).
4. After the gel’s wells have been submerged begin loading the samples.
5. Load 8-50 μL of the sample depending on the purpose of the gel.
6. Adjust the voltage to 120V.
7. Set the required time for running the gel.

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

8. Place the electrodes with the cover over the gel apparatus and initiate the electrophoresis.
9. After the time has passed turn off the machine.
10. 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

1. Take the gel out of the electrophoresis machine.
2. 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.

3. 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.
4. Afterwards follow the Thermo Scientific GeneJET Gel Extraction and DNA Cleanup Micro Kit, which is listed below as follows:
5. 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.
6. Add 200 μL of Extraction Buffer. Mix thoroughly by pipetting.
7. Incubate the gel mixture at 50-58°C for 10 minutes or until the gel slice is
completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved.
8. Add 200 μL of ethanol (96-100%) and mix by pipetting.
9. 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!


10. 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.

11. 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.

12. Repeat step 7.
13. 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.

14. Transfer the DNA Purification Micro Column into a clean 1.5 mL microcentrifuge tube (not included).
15. 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.
16. Discard the purification column and store the purified DNA at -20°C.