TITLE: Loop L2 (even level) type IIS cloning - pCs-ye vectors
AUTHORS: Eftychis Frangedakis, Susana Sauret-Gueto, Anthony West, marta tomaselli, Nicola Patron, Marius Rebmann, Jim Haseloff

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol based on</div><div class = "text-block"><a href="http://paperpile.com/b/VyRuHk/Qi6v" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Pollak B, Cerda A, Delmans M, et al (2019) Loop assembly: a simple and open system for recursive fabrication of DNA circuits. New Phytol 222:628–640</span></a><span></span></div><div class = "text-block"><a href="https://doi.org/10.1111/nph.15625" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1111/nph.15625</span></a></div></div>

[STEPS]
?. Determine DNA parts concentration with spectrophotometry (Nanodrop).
?. Prepare aliquots for DNA parts to be assembled at a concentration of 15 nM and of the pCs-pe vector at a concentration of 7.5 nM.To calculate the concentration needed for each part to assemble (not the backbone) in ng/µL, divide the length of the plasmid where they are cloned by 100. For the backbone, divide the plasmid length by 200. In this way you can prepare aliquots of your parts and backbones and add 1 µL of each one to the plasmid mix.
?. Prepare the Loop assembly Even Level reaction MM according to according to Table   AB1  Component
    Volume (μL)
  2  &nbsp;  Sterile water
    &nbsp;&nbsp;&nbsp; 2 
  3  10x Tango buffer (Thermo Fisher)
    &nbsp;&nbsp;&nbsp; 1 &nbsp;
  4&nbsp;&nbsp; 1 mg/mL bovine serum albumin
  (NEB)  &nbsp;&nbsp; 0.5 
  5  T4 DNA ligase (5 U/µL) (Thermo Fisher)
    &nbsp; 0.25 
  6  10mM ATP (SIGMA)
    &nbsp;&nbsp;&nbsp; 1 
  7  SapI (LguI) (5 U/µL) (Thermo Fisher)
    0.25 
  8  Final volume
    5
AB1  Component
    Volume (μL)
  2  &nbsp;  Sterile water
    &nbsp;&nbsp;&nbsp; 2 
  3  10x Tango buffer (Thermo Fisher)
    &nbsp;&nbsp;&nbsp; 1 &nbsp;
  4&nbsp;&nbsp; 1 mg/mL bovine serum albumin
  (NEB)  &nbsp;&nbsp; 0.5 
  5  T4 DNA ligase (5 U/µL) (Thermo Fisher)
    &nbsp; 0.25 
  6  10mM ATP (SIGMA)
    &nbsp;&nbsp;&nbsp; 1 
  7  SapI (LguI) (5 U/µL) (Thermo Fisher)
    0.25 
  8  Final volume
    5
?. Transform 20 μL of chemically competent E. coli cells (transformation efficiency of 1 × 107 transformants/µg plasmid DNA) using 2 μL of the Loop assembly reaction and then plate on LB agar plates containing 100 μg/mL spectinomycin.
?. Incubate O/N at 37 °C.
?. Colonies with white color are likely to contain the vector with the insert while yellow color colonies will contain the empty vector.
?. Confirm the presence of the correct insert with Sanger sequencing using the primers  pC_F and pC_R
?. 
?. Prepare plasmids mix for each reaction, by adding in a 0.2 mL tube, 1 μL of each DNA part aliquot (see step 2), 1 μL of the pCs-pe vector and sterile water up to 5 μL. Mix well.
?. Add 5 μL of master mix to the 5 μL of plasmids mix, to a final volume of 10 μL. Mix well. If more than 4 DNA parts are to be assembled into a pCk-pe vector, reduce the water volume in the MM by 1 μL for each extra 1 μL of DNA part added in the plasmids mix.
?. Place samples in a thermocycler and use the following program: Assembly: 26 cycles of 37​ °C for 3 min and 16 °C for 4 min. Termination and enzyme denaturation: 50 °C for 5 min and 80 °C for 10 min.
?.