McClean: Golden Gate-Making a new part: Difference between revisions

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==Protocol==
==Protocol==
# Make a 50 μL solution of BsmBI, T4 Ligase Buffer, 20 fMoles of each DNA type, and DI water.
* Make a 20 μL solution containing:
#  Digest overnight at 55°C. This overnight digest is recommended because BsmBI is not fully functional in T4 Ligase buffer. If we could, we would add NEB 3.1 buffer which is ideal for BsmBI, but we need T4 ligase buffer to make the ligation step easier.
** 20 femto moles of the entry vector pMM452 a.k.a. pYTK001.
#  Bring the solution to 1mM ATP and add T7 DNA ligase. Note that T4 Ligase buffer already included ATP, but this protocol assumes that most of it degraded overnight at 55°C. Leave at 25°C (room temp) for 1 hour. Then, cycle the solution at 42°C for 5 minutes and 16°C for 5 minutes 25 times. Then incubate it at 55°C for 1 more hour, then heat kill the enzymes at 80°C for 10 minutes.
** 20 femto moles of the DNA insert which has BsmBI cut sites
# Transform this product into competent e. Coli, and select white colonies on Chloramphenicol.
** 10 units of BsmBI, corresponding to 1 μL of NEB #R0580S containing 10,000 U/mL
** 3,000 units of T7 Ligase corresponding to 1 μL of NEB #M0318S containing 3,000,000 U/mL.
** 2 μL of 10X buffer for T4 Ligase with 10 mM ATP (NEB #B0202S). *Note* This buffer is T4 but the enzyme is T7; this is intentional. T7 has a preservative which prevents T7 from degrading at high temperatures. T4 is very similar to CutSmart buffer, plus it contains ATP.
* Bring the solution to the following temperatures in a thermocycler:
** For 30 cycles
*** 42°C for 5 minutes (cutting predominantly occurs at this temperature)
*** 16°C for 5 minutes (ligation predominantly occurs at this temperature)
** 55°C for 2 hours. This is the ideal cutting temp. It is not reached earlier to preserve the integrity of the dissolved ATP (this is my hypothesis, the manual doesn't explain why a non-ideal cutting temp is recommended.)
** 80°C for 10 minutes. This denatures the cutting and ligating enzymes.
* Transform this product into competent e. Coli, and select white colonies on Chloramphenicol. Innoculate the competent e. Coli with 0.5μL of the Golden Gate solution (~0.5 fMol of DNA). *Note: I tried adding 19.5μL of DNA and very few e. Coli grew because it was a toxic amount. I have not tried optomizing the amount of DNA to add beyond this. It may be worthwhile to innoculate 0.05μL of Golden Gate mix diluted in 9.95 μL of DI water, 0.5 μl of Golden Gate mix diluted in 9.9 μL of DI water, and 5 μL of Golden Gate mix diluted in 5 μL of DI water.


==Notes==
==Notes==
This protocol is more involved than the one recommended in the Yeast Toolkit paper. It was designed to improve the efficiency of the transformation, and the extra steps are much better than the possibility of redoing the entire experiment. The paper's instructions were designed for simplicity and speed. These instructions are designed to make it work.
This protocol is very similar to the protocol recommended in the Yeast Toolkit paper except that it recommends a 5 minute cutting period at 42 °C instead of a 2-minute cutting period, and it recommends a 2-hour cutting period at 55°C to drastically reduce the number of undigested backbone plasmids. Parts of this don't seem to make sense: we recommend the wrong cutting buffer for BsmBI, non-ideal temperatures, and excessive amounts of enzymes. The overarching reason for this is that we are attempting to both cut and ligate at in the same solution, and so we need to deviate from the optimal reaction conditions and compensate for the reduced efficiencies with excess enzymes. The burdensome cost of using excess enzymes is offset by the relative simplicity of doing the golden gate assembly in a single reaction.


*'''[[User:Cameron J. Stewart|Cameron J. Stewart]] 13:46, 15 September 2016 (EDT)''':
*'''[[User:Cameron J. Stewart|Cameron J. Stewart]] 13:46, 15 September 2016 (EDT)''':

Revision as of 07:02, 21 September 2016

Overview

This protocol describes how to insert a PCR product into the Yeast Toolkit entry vector via BsmBI digestion and T7 Ligase ligation. It is intended to supplement Lee, M. E., DeLoache, W. C., Cervantes, B. & Dueber, J. E. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synthetic Biology at <http://pubs.acs.org/doi/pdf/10.1021/sb500366v> (2015), as well as its supplementary documents.

See McClean: Designing "Yeast Toolkit" compatible primers for help making the primers to PCR amplify a Yeast Toolkit compatible part.

Materials

  • BsmBI restriction enzyme.
  • T4 Ligase Buffer
  • T7 Ligase
  • dATP
  • DNA to insert
  • Entry vector a.k.a. pYTK001 a.k.a. pMM452


Protocol

  • Make a 20 μL solution containing:
    • 20 femto moles of the entry vector pMM452 a.k.a. pYTK001.
    • 20 femto moles of the DNA insert which has BsmBI cut sites
    • 10 units of BsmBI, corresponding to 1 μL of NEB #R0580S containing 10,000 U/mL
    • 3,000 units of T7 Ligase corresponding to 1 μL of NEB #M0318S containing 3,000,000 U/mL.
    • 2 μL of 10X buffer for T4 Ligase with 10 mM ATP (NEB #B0202S). *Note* This buffer is T4 but the enzyme is T7; this is intentional. T7 has a preservative which prevents T7 from degrading at high temperatures. T4 is very similar to CutSmart buffer, plus it contains ATP.
  • Bring the solution to the following temperatures in a thermocycler:
    • For 30 cycles
      • 42°C for 5 minutes (cutting predominantly occurs at this temperature)
      • 16°C for 5 minutes (ligation predominantly occurs at this temperature)
    • 55°C for 2 hours. This is the ideal cutting temp. It is not reached earlier to preserve the integrity of the dissolved ATP (this is my hypothesis, the manual doesn't explain why a non-ideal cutting temp is recommended.)
    • 80°C for 10 minutes. This denatures the cutting and ligating enzymes.
  • Transform this product into competent e. Coli, and select white colonies on Chloramphenicol. Innoculate the competent e. Coli with 0.5μL of the Golden Gate solution (~0.5 fMol of DNA). *Note: I tried adding 19.5μL of DNA and very few e. Coli grew because it was a toxic amount. I have not tried optomizing the amount of DNA to add beyond this. It may be worthwhile to innoculate 0.05μL of Golden Gate mix diluted in 9.95 μL of DI water, 0.5 μl of Golden Gate mix diluted in 9.9 μL of DI water, and 5 μL of Golden Gate mix diluted in 5 μL of DI water.

Notes

This protocol is very similar to the protocol recommended in the Yeast Toolkit paper except that it recommends a 5 minute cutting period at 42 °C instead of a 2-minute cutting period, and it recommends a 2-hour cutting period at 55°C to drastically reduce the number of undigested backbone plasmids. Parts of this don't seem to make sense: we recommend the wrong cutting buffer for BsmBI, non-ideal temperatures, and excessive amounts of enzymes. The overarching reason for this is that we are attempting to both cut and ligate at in the same solution, and so we need to deviate from the optimal reaction conditions and compensate for the reduced efficiencies with excess enzymes. The burdensome cost of using excess enzymes is offset by the relative simplicity of doing the golden gate assembly in a single reaction.

References

Lee, M. E., DeLoache, W. C., Cervantes, B. & Dueber, J. E. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synthetic Biology at <http://pubs.acs.org/doi/pdf/10.1021/sb500366v> (2015)

Contact

or instead, discuss this protocol.


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