Team:Team:Imperial College London/Wetlab/Protocols/Cellculture/CC12

= Biobricking Parts From Oligios =

This protocol indicates how BioBricks can be assembled from oligios. If your inserts are small, oligios represent a quick, easy and cheap route to BioBricking parts. We ordered our oligios from eurofins mwg operon.

In brief, biobricking oligios involves the following steps:

1) DNA rehydration.

2) Strand Annealing.

3) Ligation.

4) Transformation.

DNA Rehydration:
DNA arrives in a dehydrated state and must be rehydrated before use. When your oligios arrive, they will come with a  Synthesis Report . This will show the total yield of DNA in micrograms.

1) Observe the DNA yield and an equal amount of sterile water such that the concentration of the resultant solution is 1 microgram of DNA per microliter of water. Pipette the water up and down to dislodge the DNA pellet from the side of the tube.

2) Leave the DNA solution to sit for 30 minutes at room temperature after which the tube should be vortexed.

Strand Annealing:
1) Take 10ul of DNA from the 1ug/ul solution and add to 90ul of sterile water. This generates a 0.1ug/ul solution.

2) Into a new eppindorf, add 8ul of annealing buffer.

3) You should have two 0.1ug/ul DNA solutions (one for each strand of your part). Add add 1ul of each of these solutions to the 8ul of annealing buffer. This gives a final 10ul solution.

4) Place the 10ul solution in a waterbath and heat to 90 degrees centigrade. This ensures that the DNA is fully melted.

5) Once the waterbath has reached 90 degrees centigrade, turn it off and allow the DNA to cool overnight in the waterbath. This results in the annealing of the two strands.

Vector Ligation:
When you design your primers, you should ensure that they have sticky ends. We designed out oligios to mimic a EcoR1 + SpeI restriction digest. This means that we can ligate them to a BioBrick vector that has been cut with these two restriction enzymes. We used the BioBrick vector AK3. With many ligations, it is common practice to dephosphorylate the vector to prevent from re-ligating in the absence of the insert. Since it is not possible to do this for oligo ligations, it is important to carry out a negative control in order to determine the background rate of vector re-ligation.

1) Prepare the following ligation mix:


 * Vector (AK3) = 0.5ul


 * Annealed oligo insert = 0.5ul


 * T4 Buffer = 1ul


 * Sterile H20 = 7ul


 * T4 Ligase = 1ul

Note, it is essential that the T4 ligase is added at the end.

2) Incubate the above mixture in a 16 degree centigrade waterbath overnight.

Transformation:
1) Pipette 1.8ul of the ligation mix into 50ul of competent cells. Pipette up and down to mix.

2) Pipette into an electro-cuvette and electroporate.

3) Add 450ul of LB broth (no antibiotic) to the electrocuvette and pipette up and down.

4) Transfer 450ul of the LB broth + cells + DNA mix into an eppindorf.

5) Incubate for 20 minutes.

6) Spin down (1 min, 10,000 rpm) pour off part of the supernatent. Resuspend cells in remaining supernatent.

7) Plate out and incubate overnight at 37 degrees centigrade.