Difference between revisions of "Team:UCLA/Notebook/Spider Silk Genetics/Protocols/Iterative Capped Assembly"

(→‎Iterative Capped Assembly)
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#*Incubate on a rotator at RT for 45 minutes.
 
#*Incubate on a rotator at RT for 45 minutes.
 
#*This incubation period can be used to prepare the subsequent ligation reactions ahead of time. Mix all the relevant reagents together EXCEPT the T7 Ligase in separate tubes, one tube per reaction.
 
#*This incubation period can be used to prepare the subsequent ligation reactions ahead of time. Mix all the relevant reagents together EXCEPT the T7 Ligase in separate tubes, one tube per reaction.
−
#Apply the magnetic strip to isolate the beads. (All future wash steps require the magnetic strip to isolate the beads)
+
#Apply the magnetic strip to isolate the beads and use a pipette to remove residual solution. (All future wash steps require the magnetic strip to isolate the beads)
 
#*Wash twice with 100 uL of 0.5x BW Buffer.
 
#*Wash twice with 100 uL of 0.5x BW Buffer.
 
#Add in the components of the first ligation:
 
#Add in the components of the first ligation:
Line 29: Line 29:
 
#Completely resuspend beads using pipet or by gently flicking.
 
#Completely resuspend beads using pipet or by gently flicking.
 
#Incubate on rotator at RT for 3 min.
 
#Incubate on rotator at RT for 3 min.
 +
#Place on magnetic rack and remove residual solution.
 +
#Wash 2x using 100 uL 0.5x BW Buffer
 
#Mix in components of second ligation:
 
#Mix in components of second ligation:
 
#*5 uL 2x T7 Ligase Buffer
 
#*5 uL 2x T7 Ligase Buffer
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#Add 0.5 uL T7 Ligase and resuspend completely.
 
#Add 0.5 uL T7 Ligase and resuspend completely.
 
#Incubate on rotator at RT for 3 min.
 
#Incubate on rotator at RT for 3 min.
 +
#Repeat wash and ligation steps as many times as needed to assemble the construct.
 +
#For the final ligation mix the following:
 +
#*5 uL 2x T7 Ligase Buffer
 +
#*1 uL of 5 uM working Terminator oligo
 +
#*1 uL of C-cap
 +
#*2.5 uL ddH2O.
 +
#Resuspend the beads and add 0.5 uL T7 Ligase Buffer.
 +
#Incubate on rotator for 3 min.
 +
#Remove residual solution.
 +
#Wash once using 100 uL 0.5x BW Buffer.
 +
#Wash once using 100 uL ddH2O.
 +
#Resuspend beads in 15 uL of [https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/Protocols/ICA_Preparation#ICA_Elution_Buffer 0.01% Tween-20].
 +
#Incubate at 95 C for 5 min.
 +
#Apply magnetic strip and save the solution in an appropriately labeled tube.

Revision as of 22:22, 29 August 2015

iGEM UCLA




This document details steps to perform ICA for Spider Silk Gene Assembly.

ICA Preparation

Iterative Capped Assembly

  1. Resuspend the M-270 Streptavidin beads by gently shaking the bottle.
  2. Aliquot 5 uL of beads into tube. Apply the magnetic strip to isolate the beads.
    • Wash twice with 100 uL of 2x BW Buffer
  3. Resuspend the beads in 5 uL 2x BW Buffer, 4 uL ddH2O, and 1 uL 50 nM Initiator.
    • Incubate on a rotator at RT for 45 minutes.
    • This incubation period can be used to prepare the subsequent ligation reactions ahead of time. Mix all the relevant reagents together EXCEPT the T7 Ligase in separate tubes, one tube per reaction.
  4. Apply the magnetic strip to isolate the beads and use a pipette to remove residual solution. (All future wash steps require the magnetic strip to isolate the beads)
    • Wash twice with 100 uL of 0.5x BW Buffer.
  5. Add in the components of the first ligation:
    • 5 uL 2x T7 Ligase Buffer
    • 50 ng of piece AB
    • ddH2O to 9.5 uL
  6. Add 0.5 uL of T7 Ligase (temperature sensitive, put back in freezer between use)
  7. Completely resuspend beads using pipet or by gently flicking.
  8. Incubate on rotator at RT for 3 min.
  9. Place on magnetic rack and remove residual solution.
  10. Wash 2x using 100 uL 0.5x BW Buffer
  11. Mix in components of second ligation:
    • 5 uL 2x T7 Ligase Buffer
    • 50 ng of piece BC
    • 1 uL of 5 uM A-cap
    • ddH2O to 9.5 uL
  12. Add 0.5 uL T7 Ligase and resuspend completely.
  13. Incubate on rotator at RT for 3 min.
  14. Repeat wash and ligation steps as many times as needed to assemble the construct.
  15. For the final ligation mix the following:
    • 5 uL 2x T7 Ligase Buffer
    • 1 uL of 5 uM working Terminator oligo
    • 1 uL of C-cap
    • 2.5 uL ddH2O.
  16. Resuspend the beads and add 0.5 uL T7 Ligase Buffer.
  17. Incubate on rotator for 3 min.
  18. Remove residual solution.
  19. Wash once using 100 uL 0.5x BW Buffer.
  20. Wash once using 100 uL ddH2O.
  21. Resuspend beads in 15 uL of 0.01% Tween-20.
  22. Incubate at 95 C for 5 min.
  23. Apply magnetic strip and save the solution in an appropriately labeled tube.