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Team:Alberta/Notebook/protocols/invitro biobyte assembly
In Vitro BioBytes Assembly 2.0
Please note that the GENOMIKON kit comes with premade anchor-Byte constructs.
The GENOMIKON kit supplies plastic micropipettes than can dispense 25ul per drop.
- Mix the iron micro beads for 10 minutes.
- Transfer a 25 ul (one drop) aliquot of iron micro beads to a 1.5 mL tube.
- Pull the beads to the side using the magnetic tube rack supplied.
- Remove and discard the supernatant.
- Add 50 ul (2 drops) of supplied Wash Buffer to the beads. Flick gently to resuspend.
- Pull the beads to the side using the magnetic tube rack.
- Remove and discard the supernatant.
- Repeat steps 5 to 7.
- Add 200 ng of a premade anchor-Byte construct to the beads and top off the volume to 25ul with the TE Buffer supplied. Flick gently to resuspend.
- Allow annealing for 30 minutes, mixing by flicking every 5 minutes. Ensure that there are no droplets on the sides of the tube.
- Repeat steps 6 and 7.
- Repeat steps 5 to 7.
- Add 200 ng of the next Byte of your construct, making sure that a BA Byte follows an AB Byte and vice versa. Add an appropriate amount of 2x QuickLigase Buffer, Quick Ligase and TE to a total volume of 25ul.
- Example:
| Reagent | Volume (ul) |
|---|---|
| (40ng/ul) AB kan Byte | 5 |
| 2X Quick Ligase Buffer | 13 |
| TE Buffer | 6 |
| Quick Ligase | 1 |
| TOTAL | 25 |
- 14. Flick gently to resuspend. Allow ligation for five minutes, flicking gently every minute.
- 15. Add 25 ul of Wash Buffer to the tube. Flick gently.
- 16. Repeats steps 6 and 7.
- 17. Repeat steps 5 to 7 twice.
- 18. Repeat steps 13 to 17 for each subsequent Byte addition, including the last Byte. If the last Byte is cap, it must be added in 20 X molar excess.
- 19. After the last Byte has been ligated and washed, addition of 25 ul of 75C Elution Buffer. The entire tube should be kept at 75C for ten minutes to allow the DNA construct to elute off the beads.
- 20. After ten minutes, put the tube into a magnetic rack in the 75C waterbath. Allow the beads to be pulled aside and collect the supernatant into a clean 1.5 mL tube. The supernatant will contain your construct.
