Nextera/MiSeq Plasmid Validation

Turnaround time

As low as 3 business days (depending on sample numbers and queue status).

We also accept priority samples. Users will get results in 3 business days for up to 250 samples.

User Guidance

Sample Preparation

• Nextera/MiSeq sequencing is sensitive to contamination. To avoid contamination issues during sample preparation, please do the following:

  • Use nuclease-Free Water, Molecular Biology Grade P/N NUPW-0050 (Growcells brand). 

  • Use DNA AWAY Surface Decontaminant P/N 7010, and RNaseZap Decontamination Solution P/N AM9782 (available in the ESE main stockroom) to clean your bench and pipettes during sample preparation. 

• Researchers can submit samples as purified plasmids, PCR amplicons, or bacterial cultures.

  • Purified plasmids or PCR amplicons:

    • Quantify each sample by the Qubit HS DNA Assay. 

    • Purified DNA should be in nuclease-free water or 10 mM Tris-Cl, pH 8.5 (Qiagen Elution Buffer); do NOT use EDTA  (e.g., no TE buffer) as this inhibits Nextera enzymes.

    • Samples should be diluted to 0.1 ng/uL concentration, in 40 uL volume (4 ng total).

(Please submit the concentration and volume indicated above, to be compatible with our automation processes. )

• For PCR amplicons:

  • You must purify or remove primers (e.g. Exo-CIP™ Rapid PCR Cleanup Kit; link). Otherwise primer/primer dimer carryover could take up a significant proportion of sequencing reads on the MiSeq (since smaller templates cluster preferentially on MiSeq flow cells)

  • The minimum size is 1 kb.  It is recommended that the original PCR was performed with primers at least 50 bases upstream/downstream of your target region of interest because Nextera enzymes cannot bind to the distal ends of DNA fragments. 

• Bacterial cultures(1 plasmid type per E coli culture. NO yeast cultures):

  • Cell cultures are accepted only for high-copy plasmids . 

  • There should be visible cell growth for submission (OD600 ≥0.3).

  • Dilute 20 uL E. coli cell culture with 20 uL nuclease free water, and boil at 95 C for 5 minutes.  Then submit 40 uL of boiled culture.

• Submit samples in an Echo Qualified 384-Well Polypropylene Microplate (384PP v2.0), regardless of sample number or type. These Echo plates are available in the ESE SB3 satellite stockroom PP-0200. 

• Label the plate with your lab username, plate number, and the date (YYYYMMDD), e.g. “NJHillson_1_20221010”. Well A1 should be at the top left corner on the plate when you face your label. 

• Seal the plate securely to avoid cross-contamination, but do not use heat sealers. 

  • It is recommended to use the Bio-Rad foil seals, Microseal 'F' PCR  MSF1001, which are available in the ESE main stockroom.

• Deliver samples to FRZ-0307 4D, in 4203 (across from the Robotics lab, near the warm rooms).

• The deadline for sample submission is at 3:00 pm on Friday