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.