Direct sequencing of bacterial colonies by rolling circle amplification (RCA)
The Direct Colony Sequencing service uses rolling circle amplification to enable fast sequencing of bacterial colonies in 96well plates without the need for plasmid preparation.
Fast
Receive your sequencing results one day earlier for your plasmid clones as DNA preparation is not needed. Results are available 15 hours after samples received.
Convenient
With our PlateSeq Kit Colony you get all the accessories you need to ship your bacterial colonies either from agar colonies or as liquid culture. Use your nearest DropBox for free shipping or our Shipping Kits if no DropBox is available.
Flexible
You can choose between a prepaid option using our PlateSeq Kit Colony and a non-prepaid option with our ColonySeq Plate. To prepay for additional reactions you can use our Plate Coupons. Up to 4 reactions per sample can be specified.
Cost-Effective
Reduce your expenses on plasmid DNA isolation and let us sequence your bacterial colonies directly.
Please follow the instructions in our sample submission guide how to prepare and send your samples.
Specifications of our Direct Colony Sequencing service:
- Available for E.coli colonies or any other gram negative bacteria
- Up to 4 reactions per sample, per plate are possible
- High quality read lengths of up to 1,100 bases
- Results are available 15 hours after samples received
- Free one time repetition in case of technical failure*
- Different sequencing result files can be selected per order
- A hugh amount of standard primers are available free of charge
- Sequencing primers can be ordered or enclosed
- Ordered primers are stored for 4 weeks
- Primer storage for one year, on request
- Secure online acccess to your sequence data for 6 months
Sending samples the right way for our Direct Colony Sequencing Service
Sample preparation and submission
- Please order either our PlateSeq Kit Colony or the ColonySeq Plate first (They contain the approptiate buffer)
- Shortly centrifuge the received plate to avoid the liquid on the foil. Carefully remove the sealing foil from the plate
- Grow your colonies on agar plates long enough to have a colony diameter of at least 1 mm*
- Use a toothpick to take as much of the bacterial colony as possible and inoculate into our plates. Swirl the toothpick for some seconds to transfer as many cells as possible into the wells
- In case of bacterial suspension use a pipette to add 5µl of the liquid culture in each well
- Well H12 should be kept empty for internal quality control
- Seal your plates using 8-cap strips to prevent material loss (8-cap strips are provided along with each PlateSeq Kit Colony)
- Plates can be sent / provided at ambient temperature to our sequencing lab
Sequencing primers
You can either choose from our list of available standard primers, re-use one of your stored primers, order the primer synthesis from us or enclose your own primers in tubes or as separate primer plate mirrored to the sample plate. Do not premix primers with your templates!
Optimum primer conditions:
- Primers must not contain phosphorylation or fluorescent dyes
- The optimum primer length is between 16-25 bases
- The primer melting temperature (Tm) should be 50 - 62°C
- The GC content of the primer should be 35-60%
- Ideally one G or C should be located at the 3' primer end
- The number of 3' Gs or Cs should not exceed 2 Gs or Cs
- If possible, avoid >3 identical bases in a row in the sequence
Primer concentration and volume:
- Exactly 10 pmol/µl primer concentration is required per sequencing reaction
- Each primer must have a total volume of 15 µl (double distilled water or 5mM Tris-HCl); 5 µl of primer volume is required for every additional sequencing reaction
- Concentration of primers with wobble bases must be calculated according to the following formula: nX x ConcPrimer