Team:Arizona State/Lab/Protocols/Assembly

Poly-X Ligation

The goal of this protocol is to produce a polymer of DNA starting from a single unit with the Biobrick prefix and suffix. Cutting with XbaI and SpeI produces sites that can ligate into XbaI-XbaI, XbaI-SpeI, or SpeI-SpeI. Finally, the ligation product can be cut with XbaI and SpeI a final time. XbaI-SpeI scars are not cut, while any incorrectly oriented inserts are. This allows the DNA to form chains of various lengths. See results for further characterization of this process.

File:Arizona State poly-x

Procedure

1. Run a PCR of your BioBrick (ideally this PCR should not produce extra bands, as these may eventually interfere with the ligation).
2. Following the PCR, use the solution as the DNA sample for restriction digest.

   Restriction reaction mixture:

   50µL PCR product

   7µL 10x NEB4 buffer

   8µL H₂0

   1µL BSA (10mg/ml)

   2µL XbaI

   2µL SpeI

3. Incubate at 37C for at least 1 hour.
4. Column purify the 70µl restriction reaction by following Sigma-Aldrich column purification protocol. For the elution step use 50µl elution solution.
5. From the column purified product: use 25µl for ligation and keep 25µl for a diagnostic gel and/or future use.

   Ligation reaction mixture:

   25µl column prepped DNA

   17µl H₂0

   5µl 10x T4 ligase buffer

   3µl T4 DNA ligase

   Continue ligation for at least 1 hour at room temperature. I recommend 12-16 hours at 16C when larger linear products are desired.

6. If orientation of the product is a concern, run a final XbaI + SpeI restriction to eliminate X-X and S-S sites.
7. Following the previous step a small portion (<10µl ligation reaction) should be visualized by a 1 or 2% agarose gel. About 5µl of column preparation product and/or restriction product should be run in the adjacent lane to the ligation product.
8. Run the gel and assay the poly-X product created (e.g. 1x, 2x, 3x etc.). I don't recommend gel extraction, it may help in isolating a specific polymer length, so you have to determine if you have a high enough concentration to extract (GEL EXTRACT AT YOUR OWN RISK).
9. Take an aliquot of ligation product, and run in a new ligation reaction with pSB1A3, or 1K3 etc. Cut plasmid with XbaI and SpeI (or whatever restriction enzymes necessary) and dephosphorylate using the standard protocols. (I recommend using higher concentrations of backbone than the protocol usually calls for). I recommend running a ligation of linearized plasmid only, this will serve as a negative control, where if you get results either the plasmid wasn't completely cut or dephosphorylated.

   NOTE: For mini-prepped plasmids it is always a commendable effort to run cut (linearized) and uncut (unlinearized) on an agarose gel to determine the purity of the sample. Simply looking at nano-drop results and 'scoring high' is meaningless because you may have contamination, however when using the nanodrop to good things to note are 230:260:280 which should be 1: 1.8: 1 respectively. Jagged peaks mean you need to re-blank the instrument and clean the pedestal.

10. Ligate the poly-X sample with the linearized vector using the basic ligation protocol (Consider what ratios would be best for the ligation). Also consider elongated ligation time (to long of ligation can reduce transformation efficiency due to long linear products).
11. Transform the product with the necessary controls.

Ginkgo

Biobrick