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

(→‎ICA Considerations)
 
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#Resuspend the M-270 Streptavidin beads by gently shaking the bottle.  
 
#Resuspend the M-270 Streptavidin beads by gently shaking the bottle.  
 
#Aliquot 5 uL of beads into tube. Apply the magnetic strip to isolate the beads.
 
#Aliquot 5 uL of beads into tube. Apply the magnetic strip to isolate the beads.
−
#*Wash twice with 100 uL of 2x BW Buffer
+
#*Wash twice with 100 uL of [https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/Protocols/ICA_Preparation#2x_Binding_and_Wash_.28BW.29_Buffer 2x BW Buffer]
 
#Resuspend the beads in 5 uL 2x BW Buffer, 4 uL ddH2O, and 1 uL 50 nM Initiator.
 
#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.
 
#*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:
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#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
Line 36: Line 38:
 
#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 ICA Elution Buffer].
 +
#Incubate at 95 C for 5 min.
 +
#Apply magnetic strip and save the solution in an appropriately labeled tube.
 +
 +
==ICA Considerations==
 +
*The part collection we designed for ICA with spider silk genes contains two parts indicated as MaSp1 SeqAB and MaSp2 SeqAB. These parts have a different DNA sequence than the other MaSp1 and 2 pieces respectively, but code for the same protein. These parts assist in sequencing long ICA constructs.
 +
*See our primers page to find primers that may be used to sequence from the sequencing cores.
 +
 +
=ICA Product Amplification and cloning=
 +
*Use one microliter of the ICA eluate as template for PCR amplification using [https://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics/ICA_Oligo_Sequences post-elution primers].
 +
*The amplified product can be purified and cloned into the vector of choice.

Latest revision as of 17:15, 18 September 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.
  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 ICA Elution Buffer.
  22. Incubate at 95 C for 5 min.
  23. Apply magnetic strip and save the solution in an appropriately labeled tube.

ICA Considerations

  • The part collection we designed for ICA with spider silk genes contains two parts indicated as MaSp1 SeqAB and MaSp2 SeqAB. These parts have a different DNA sequence than the other MaSp1 and 2 pieces respectively, but code for the same protein. These parts assist in sequencing long ICA constructs.
  • See our primers page to find primers that may be used to sequence from the sequencing cores.

ICA Product Amplification and cloning

  • Use one microliter of the ICA eluate as template for PCR amplification using post-elution primers.
  • The amplified product can be purified and cloned into the vector of choice.