Basically, do exactly the same as Add barcodes during PCR (public copy) but with a different assembly step so we don’t have cloning issues, using a large-site restriction enzyme.
The problem with First time: fragment genomes (public copy) was that the AarI restriction enzyme cut many of the genome inserts, reducing our library size and biasing against bigger inserts. One way around this is with other restriction sites that target much larger pieces of DNA. Specifically, I want to use the enzyme I-CeuI https://www.neb.com/en-us/products/r0699-i-ceui, which has the following 27-bp recognition site. That should work.
Therefore, the overall goal is to:
Clone barcoded metagenomic library @c.00214.BsT-mStayGold_bboneBarcode-MegaNuclease-Helongata : tagmentation rxn (HE gDNA w/meganuclease restriction site) + barcoded plasmid backbone (w/ meganuclease restriction site. )
The goal is to make plasmid using the barcoding and tagmentation strategy below:
PREPPING BARCODED PLASMID BACKBONE:
| Input DNA | PCR# | F primer | R primer | Extension time | Tm |
|---|---|---|---|---|---|
| @c.00185.pTwist_BsT-mStayGold_Barcode-GG | 346 | @p00103 | @p00104 | 90s | 65 |
TAGMENTATION:
RESTRICTION DIGESTION & LIGATION
After PCR, the PCR product must be purified, digested according to the I-CeuI protocol, then purified again.
The same must be done after tagmentation - purification, digestion, purification.
Then both digested pieces of DNA are ligated together, following the instructions of the ligation kit