454 GS FLX+ Template Preparation Instructions

  GS FLX Titanium Sample Preparation    

Shotgun sequencing projects:

  • DNA must be double-stranded
  • Minimum of 500 ng of high quality, high molecular weight, RNA-free DNA
  • DNA concentration should be at least 25 ng/µl in Qiagen elution buffer (EB) or low TE buffer (10 mM Tris-HCl, pH7.5-8.0; 0.1 mM EDTA)
  • DNA must be quantified using a fluorometer.  Spectrophotometry and Nanodrop will often give readings that are greatly overestimated.
  • DNA should not contain any particulate matter

Paired-end (PE) sequencing projects:

  • DNA must be double-stranded
  • Input DNA should be >10 kb
  • Minimum of 5 µg of DNA is required for 3 kb PE library
  • Minimum of 10 µg of DNA is required for 8 kb PE library
  • DNA concentration should be a minimum of 25 ng/µl in EB or low TE buffer
  • DNA must be quantified using a fluorometer.  Spectrophotometry and Nanodrop will often give readings that are greatly overestimated.
  • DNA should not contain any particulate matter

Amplicon sequencing projects:

  • DNA must be double-stranded
  • Minimum of 20 ng of DNA in a 10-20 µl volume.
  • PCR clean-up method should be described (if desired, AGTC can perform post‑PCR clean-up)
  • DNA must be quantified using a fluorometer.  Spectrophotometry and Nanodrop will often give readings that are greatly overestimated.
  • DNA should not contain any particulate matter
  • Optional:  client can incorporate A and B adaptors into their gene-specific PCR primers:

o    A: 5’-CGTATCGCCTCCCTCGCGCCATCAG-MID-template specific sequence-3’

o    B: 5’-CTATGCGCCTTGCCAGCCCGCTCAG-MID-template specific sequence-3’

(underlined sequence is an essential key, the MID is a multiplex identifier sequence which allows pooling of amplicons from different individuals – length depends on multiplicity needed).

RNA-seq projects:   

  • Minimum of 25 µg high quality total RNA (if AGTC will isolate mRNA)
  • Minimum of 250 ng mRNA
  • Minimum of 500 ng rRNA-depleted total RNA RNA must arrive on dry ice

Customer Quality Assurance:

Any gel pictures, Agilent traces, OD 260/280, or fluorescent quants which the customer has performed should be forwarded to AGTC.