TITLE: Loop L2 (even level) SapI type IIS cloning into pCs 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]
?. [Protocol for assembly of L1 constructs into a L2 device]
Determine the concentration of each DNA plasmid needed (L1 plasmids and pCs acceptor plasmid)with spectrophotometry (Nanodrop).In the example in step 2, determine concentration of plasmids L1_001-Ck1, L1_002-Ck2, L1_003-Ck3, L1_004-Ck4 and pCsA
?. [Protocol for assembly of L1 constructs into a L2 device]
Prepare aliquots for each plasmid at a concentration of 15 nM for the L1 plasmids and of 7.5 nM for the acceptor pCs vector. With this final concentration, 1 µL of each plasmid is added to the plasmids mix (see step 6).To calculate the concentration in ng/μL: -  For a final concentration of 15 nM, the concentration in [ng/ul]  equals N (the length in bp of the plasmid) divided by 110. This is an approximation of the formula:15∙10^(-9)mol/L x ((607.4 x N ) + 157.9)g/mol x 10^(-6)L/μL x 10^9ng/g = concentration (ng/μL)- For a final concentration of 7.5 nM, the concentration in [ng/ul]  equals N divided by 220.
?. [Protocol for assembly of L1 constructs into a L2 device]
Prepare the Loop assembly Even Level reaction master mix (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
?. [Protocol for assembly of L1 constructs into a L2 device]
Prepare plasmids mix by adding in a 0.2 mL tube: 1 μL of each of the 4 L1 plasmids , and 1 μL of the pCs (see step 2). Mix well.
?. [Protocol for assembly of L1 constructs into a L2 device]
Add 5 μL of master mix (step 5) to the 5 μL of plasmids mix (step 6), to a final volume of 10 μL. Mix well.
?. [Protocol for assembly of L1 constructs into a L2 device]
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.
?. [Protocol for assembly of L1 constructs into a L2 device]
Transform 20 μL of chemically competent E. coli cells (transformation efficiency of 1 × 10^7 transformants/µg plasmid DNA) using 2 μL of the Loop assembly reaction and then plate on LB agar plates containing 100 μg/mL spectinomycin and 40 μg/mL of X-gal for blue-white screening.
?. [Protocol for assembly of L1 constructs into a L2 device]
Incubate O/N at 37 °C.
?. [Protocol for assembly of L1 constructs into a L2 device]
Colonies with white color are likely to contain a L2 insert cloned into the pCs vector (In the example in step 2: 001-002-003-004)Blue color colonies will contain undigested pCs vector with LacZ
?. [Protocol for assembly of L1 constructs into a L2 device]
Confirm the presence of the correct insert with Sanger sequencing using the primers  pC_F (GCAACGCTCTGTCATCGTTAC) and pC_R (GTAACTTAGGACTTGTGCGACATGTC) for pCs vectors, and pC_F and pC_R2 (CAATCTGCTCTGATGCCGCATAGTTAAG) for pCschlo vectors.
?. [Loop pCs vectors]
?. [Example of assembly of L1 constructs into a L2 device]