TITLE: Loop L1 (odd level) BsaI type IIS cloning into pCk vectors
AUTHORS: Eftychis Frangedakis, Susana Sauret-Gueto, Anthony West, Nicola Patron, marta tomaselli, 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]
?. [Loop pCk vectors]
?. [Example of assembly of L0 parts into a transcription unit (L1)]
?. [Protocol for assembly of L0 parts into a transcription unit (L1)]
Determine the concentrations of each DNA plasmid needed (L0 plasmids and pCk acceptor plasmid) by spectrophotometry (Nanodrop).In the example in step 2, determine concentration of plasmids L0_PROM5, L0_CDS, L0_3TERM and pCk1.
?. [Protocol for assembly of L0 parts into a transcription unit (L1)]
Prepare aliquots for each plasmid at a concentration of 15 nM for the L0 plasmids and of 7.5 nM for the acceptor pCk 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 L0 parts into a transcription unit (L1)]
Prepare Loop assembly Level 1 reaction master mix (MM) according to Table , if four or less number of L0 parts are assembled into a pCk vector (otherwise see step 8)  AB1  Components
    Volume (μL)
  2  Sterile water 
    3 
  3  10x T4 ligase buffer (NEB)
    1 
  4  1 mg/mL bovine serum albumin (NEB)
    0.5 
  5  T4 DNA ligase at 400 U/μL (NEB)
    0.25 
  6  10 U/μL BsaI (NEB)
    0.25 
  7  Final volume
    &nbsp;5
AB1  Components
    Volume (μL)
  2  Sterile water 
    3 
  3  10x T4 ligase buffer (NEB)
    1 
  4  1 mg/mL bovine serum albumin (NEB)
    0.5 
  5  T4 DNA ligase at 400 U/μL (NEB)
    0.25 
  6  10 U/μL BsaI (NEB)
    0.25 
  7  Final volume
    &nbsp;5
?. [Protocol for assembly of L0 parts into a transcription unit (L1)]
Prepare plasmids mix by adding in a 0.2 mL tube: 1 μL of each L0 plasmid , 1 μL of the pCk vector (see step 4), and sterile water up to 5 μL. Mix well.When 4 L0 parts are assembled into a pCk plasmid, the volume of the plasmid mix is 5 μL, and thus no volume of water is added.
?. [Protocol for assembly of L0 parts into a transcription unit (L1)]
Add 5 μL of MM (step 5) to the 5 μL of plasmids mix (step 6), to a final volume of 10 μL. Mix well.
?. [Protocol for assembly of L0 parts into a transcription unit (L1)]
If more than 4 L0 parts are to be assembled into a pCk vector, reduce the water volume in the MM by 1 μL (step 5) for each extra 1 μL of DNA part added in the plasmids mix  (step 6).
?. [Protocol for assembly of L0 parts into a transcription unit (L1)]
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 L0 parts into a transcription unit (L1)]
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 50 μg/mL kanamycin and 40 μg/mL of X-gal for blue-white screening.
?. [Protocol for assembly of L0 parts into a transcription unit (L1)]
Incubate overnight at 37 °C.
?. [Protocol for assembly of L0 parts into a transcription unit (L1)]
Colonies with white color are likely to contain an L1 insert cloned into the pCk vector (In the example in step 2: PROM5:CDS-3TERM)Blue color colonies will contain undigested pCk vector with LacZ
?. [Protocol for assembly of L0 parts into a transcription unit (L1)]
Confirm the presence of the correct insert with Sanger sequencing using the primers  pC_F (GCAACGCTCTGTCATCGTTAC) and pC_R (GTAACTTAGGACTTGTGCGACATGTC) for pCk vectors, and pC_F and pC_R2 (CAATCTGCTCTGATGCCGCATAGTTAAG) for pCkchlo vectors.