Team:Alberta/Notebook/protocols/invitro biobyte assembly

From 2010.igem.org

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===Reagents:===
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===Please note that the GENOMIKON kit comes with premade anchor-Byte constructs.===
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===The GENOMIKON kit supplies plastic micropipettes than can dispense 25ul per drop.===
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* 1.5mL Eppendorf tubes
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:# Mix the iron micro beads for 10 minutes.
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* Magnet
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:# Transfer a 25 ul (one drop) aliquot of iron micro beads to a 1.5 mL tube.
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* Wash/binding buffer (10mM Tris 1mM EDTA pH8.0)
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:# Pull the beads to the side using the magnetic tube rack supplied.
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* Elution buffer ?
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:# Remove and discard the supernatant.
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* 5x ligase buffer
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:# Add 50 ul (2 drops) of supplied Wash Buffer to the beads. Flick gently to resuspend.
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* Ligase
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:# Pull the beads to the side using the magnetic tube rack.
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* PCR cleanup kit
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:# Remove and discard the supernatant.
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* Para magnetic beads (oligo-dT25mer NEB# S1419S)
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:# Repeat steps 5 to 7.  
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* A18_AB anchor stock solution (0.1pM; 67ng/uL in TE)
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:# 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.
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* AB KanR byte @ 40 ng/uL (0.06 pM/uL; gel purified in E buffer; 0.9 kbp)
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:# Allow annealing for 30 minutes, mixing by flicking every 5 minutes. Ensure that there are no droplets on the sides of the tube.
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* BA Byte (0.1pM; 67ng/uL in TE)
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:# Repeat steps 6 and 7.
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:# Repeat steps 5 to 7.
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:# 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.
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===Procedure:===
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::Example:
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{|style="margin-left:40px;"
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Preparing AB byte Anchor:
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! Reagent !! Volume (ul)
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*Add in a reaction:
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{|
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|KanR AB Byte (2.2ug; 4pM) || 5uL
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|-
|-
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|Anchor (900 ng; 50pM) || 4uL
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| (40ng/ul) AB kan Byte || 5
|-
|-
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|Q-Ligase buffer (x2) || 20uL
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| 2X Quick Ligase Buffer || 13
|-
|-
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|Q-ligase || 1uL
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| TE Buffer || 6
|-
|-
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|Total || 40uL
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| Quick Ligase || 1
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|-
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| TOTAL || 25
|}
|}
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*5 minutes @ R/T followed by heat inactivation @65<sup>o</sup>C for 10 minutes.
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::14. Flick gently to resuspend.  Allow ligation for five minutes, flicking gently every minute.
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::15. Add 25 ul of Wash Buffer to the tube. Flick gently.
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===Binding:===
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::16. Repeats steps 6 and 7.
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* Mix beads with a couple of shakes followed by 10 minutes slow rotation.
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::17. Repeat steps 5 to 7 twice.
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* Wash x2 with 50uL TE buffer
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* Add anchor byte ((0.4pM;0.27ug) and top to 20uL with TE.
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* 30 minutes of repeated flicking and inversion
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* 2x Wash as above
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===Ligation:===
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* Add:
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{|
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|MilliQ water || 6uL
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|-
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|BA Byte (0.4pM;0.27ug total) || 4uL
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|-
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|2x Q-ligase buffer || 10uL
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|-
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|Q-ligase || 1uL
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|-
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|Total || 20uL
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|}
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* 5 minutes @ R/T with gentle mixing.
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* 2x Wash as above
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::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. 
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===Elution:===
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::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.
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* Add 20uL of elution buffer @70<sup>o</sup>C.
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::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.
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* Mix and remove rapidly.
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{{Team:Alberta/endMainContent}}
{{Team:Alberta/endMainContent}}

Latest revision as of 03:49, 28 October 2010

TEAM ALBERTA


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.

  1. Mix the iron micro beads for 10 minutes.
  2. Transfer a 25 ul (one drop) aliquot of iron micro beads to a 1.5 mL tube.
  3. Pull the beads to the side using the magnetic tube rack supplied.
  4. Remove and discard the supernatant.
  5. Add 50 ul (2 drops) of supplied Wash Buffer to the beads. Flick gently to resuspend.
  6. Pull the beads to the side using the magnetic tube rack.
  7. Remove and discard the supernatant.
  8. Repeat steps 5 to 7.
  9. 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.
  10. Allow annealing for 30 minutes, mixing by flicking every 5 minutes. Ensure that there are no droplets on the sides of the tube.
  11. Repeat steps 6 and 7.
  12. Repeat steps 5 to 7.
  13. 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.


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