MGH   
CCIB
 
Sanger DNA Sequencing: Sample Submission

 

Sample Preparation (Pre-Mixed):

For sequencing plasmids or purified PCR amplicons, please provide DNA template and primer premixed!

  • In the same tube (or plate well), mix DNA template (10 µl) and sequencing primer (10 µl) according to the table below:

    DNA Template Primer
    Type Size Concentration Volume Total Mass * Concentration Volume
    double-stranded Plasmid < 10 kb 100 -200 ng/µl 10 µl ~ 1-2 µg 1.5-3.0 µM 10 µl
    double-stranded Plasmid > 10 kb 200 ng/µl 10 µl ~ 2 µg 1.5-3.0 µM 10 µl
    Cosmids/ Fosmids ~40 kb 400 ng/µl 10 µl ~ 4 µg 1.5-3.0 µM 10 µl
    Single-stranded Phage DNA 1-9 kb 100 ng/µl 10 µl ~1 µg 1.5-3.0 µM 10 µl
    PCR Product < 0.5 kb 10-20 ng/µl 10 µl ~ 200-300 ng 1.5-3.0 µM 10 µl
    PCR Product 0.5 - 1kb 20-50 ng/µl 10 µl ~ 300-500 ng 1.5-3.0 µM 10 µl
    PCR Product > 1kb 50-100 ng/µl 10 µl ~ 0.5-1 µg 1.5-3.0 µM 10 µl

    *Primer Concentration:
    Compared to our old webpage where we have specified the mass concentration of the primer solution, we are now referring to the corresponding molar concentration. We have not changed the sample preparation requirements! For a sequencing primer with a length of 20 bases, a molar concentration of 1.5 µM (1.5 pmole/µl) corresponds to a mass concentration of 10 ng/µl.

    IMPORTANT:
    We typically request a larger sample volume than we need. The submitted sample volume of 20 µl is sufficient to perform multiple reactions and allows for any repeats that we may need to perform. If your template amount should be limited for any reason, you may submit a reduced sample volume. Please contact us for discussing available options.

    For optimum results with automated fluorescent sequencing, DNA template of sufficient quality and quantity should match our guidelines (as listed in the table above and described in the Template Preparation section). If you find that your samples do not meet our requirements, please contact us so that we can assist you in identifying a potential solution.

    Equally important for the success of a sequencing reaction is the use of high-quality sequencing primers. Please refer to our Primer Design guidelines.

  • Please prepare samples in 1.5 ml tubes or a 96-well plate according to our specifications (see below).
    Please note: We have discounted pricing available for full plate (bulk) orders (only when submitting 95 samples per plate). (only when submitting 95 samples per plate).

  • Place your Sanger DNA Sequencing Order online and include a printed copy of the barcoded order form with your sample submission.

 

Tube Orders:

  • If you are submitting your samples in tube format, please use standard 1.5 ml flip-cap Eppendorf-type tubes.
    Please note: Our operational setup does not accept 0.2 ml tubes, 0.5 ml tubes, strip-tubes, and screw-cap tubes.

  • Please label the top of the lid of each tube - as clearly as possible - with a maximum of 5 alpha-numeric characters (for example: 5B001). We strongly recommend to hand-write each label using a permanent marker. Self-adherent stickers might fall off during shipment, thereby making sample identification impossible.

  • Tubes should be submitted in order as listed on the submission form (not randomly placed, and not loose/unracked).
    Please note: If you are using one of our remote drop-off locations, please rack your tubes in one of the provided sample boxes and attach order form with a rubber band.
    If for some reason no boxes should be available, please tape tubes to the order form (or a separately attached sheet) in the same order as listed on your order form. You may also use any other type of rack/box designed to hold 1.5 ml tubes.

 

Plate Orders

  • If you are submitting your samples in plate format, please use only V-bottom PCR plates.

  • We recommend the use of semi-skirted (or skirted) plates to prevent the PCR plate from bending during transit, which could result in loosening of the seal and subsequent sample loss and cross-contamination.

  • Please arrange your samples horizontally (in rows) in the PCR plate. Proceed from well A1 to A12, from B1 to B12, from C1 to C12, etc.

  • We accept partial plates but please do not leave any empty wells between samples, and please do not leave any empty spaces between samples on the order form.

    IMPORTANT:
    The maximum allowable number of samples per plate is 95. Well H12 MUST be left empty for our internal process control!
    Please note: The discounted bulk rate applies for full plates only!

  • The order form must match the order and position of samples in the PCR plate.

  • Please label the plate as clearly as possible - with a maximum of 5 alpha-numeric characters (for example: 5B001).

  • Tightly seal your plate(s) with an adhesive sheet/foil to prevent samples from drying and/or cross-contamination.

 

Sample Shipment:

Please submit your sample(s), including a hard copy of the completed order form, by using one of our remote dropboxes (please see here) or by delivering it personally to our core facility.

If remote or direct sample drop-off should not be an option for you, please ship your samples, including a hard copy of the completed order form, via a courier delivery service of your choice (for example, overnight shipment via FEDEX, UPS, etc.) to our core facility. Samples can be shipped at ambient temperature.

Shipping Tubes:

Unfortunately, we often receive sample tubes that were damaged during shipment (cracked or shattered tube, sample loss due to leakage or evaporation). Therefore, we strongly recommend sealing the sample tube with Parafilm and further protecting it with bubble wrap or several layers of Kimwipe tissue. Another option is to place the sample tube into a secondary container such as a Falcon tube or small cardboard or plastic box.

Shipping 96-well Plates:

Samples should be arrayed in a 96-well (semi-skirted or skirted) plate. To prevent sample loss and/or cross contamination, we recommend tightly sealing all wells of the plate with Microtube Caps. PCR Tube Strip Flat Caps (Eppendorf cat# 0030124847 or VWR cat# 75874-710) work well for most plates. Our everyday experience tells us that plate foils/plastic seals can partially lift off or even entirely detach during transit dependent on the shipping conditions (heat, air pressure, humidity, etc.). Please place your plate in a corrugated box for maximum protection.

Shipping Address:


MGH CCIB DNA Core Facility
38 Sidney Street / Suite 100
Cambridge, MA 02139
U.S.A.

IMPORTANT: Please note that we are not receiving samples on Saturdays and Sundays or on official holidays.