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

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=<b> Biobricking Parts From Oligios </b>=
=<b> Biobricking Parts From Oligios </b>=
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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 <i>eurofins mwg operon</i>.
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In brief, biobricking oligios involves the following steps:
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1) DNA rehydration.
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2) Strand Annealing.
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3) Ligation.
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4) Transformation.
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==<b>DNA Rehydration:</b>==
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DNA arrives in a dehydrated state and must be rehydrated before use. When your oligios arrive, they will come with a <i> Synthesis Report </i>. This will show the total yield of DNA in micrograms.
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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.
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2) Leave the DNA solution to sit for 30 minutes at room temperature after which the tube should be vortexed.
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==<b>Strand Annealing:</b>==
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1) Take 10ul of DNA from the 1ug/ul solution and add to 90ul of sterile water. This generates a 0.1ug/ul solution.
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2) Into a new eppindorf, add 8ul of annealing buffer.
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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.
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4) Place the 10ul solution in a waterbath and heat to 90 degrees centigrade. This ensures that the DNA is fully melted.
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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.

Revision as of 11:24, 29 September 2009

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.

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