SOP for High Throughput Sequencing -- Microtiter plates

All High Throughput Sequencing projects need prior approval before
submitting samples. Protocols for HTSeq are different and much more
critical. If you have never sent a HTSeq request, call Lynn X-6535 or
Claudia X-5924 before submitting samples.

# The Paper Work

> Customer must make LIMS request and include a hard copy of the Request

Details page with their samples.

> Sample and request labels must be UNIX compatible text. This is
>
> alphanumeric, dot, dash, underscore ONLY. **NO SPACES!**
>
> Position H12 is reserved for the LMT control. Do not enter or upload
> data for this
>
> position. Do not put DNA in the H12 position of any plate.

# Samples Preparation

> Samples must be submitted in 96-well(PE part #N801-0560) or
> 384-well(Griener part #785201) microtiter plates. Seal with foil seals
> (Beckman part #538619). Plastic seals allow excessive evaporation.

**For Sequencing**

> Wells must contain Template and Primer in the following proportions:

Primer 0.5 uM 2.2ul

Template 125ng/ul plasmid or 5.5ul

[50-100ng/ul PCR]{.underline}

Total 7.7ul

**For G50 Purification and Sequencing**

Plasmid DNA 125-250ng/ul 10ul

A separate primer plate must be provided or a stock primer plate must be

specified for sequencing.

# Shipping

Samples must be packaged and shipped according to NIH guidelines.

For our Frederick customers to ship sequencing samples to Tollhouse
Ave.:

A.  Safety requires a "Request for Shipment" form be filled out and sent
    to them prior to shipping any samples via the courier. A one time
    blanket form can be set-up for sending sequencing samples to
    Tollhouse Ave. which will streamline your paperwork. You can contact
    Sue Smith in Safety 301-846-5919 for details on this process.

B.  Call the courier by 4:30 pm the day before you plan on sending your
    samples. Let them know that you will have samples shipping to
    Tollhouse Ave. the next morning.

C.  Package your samples in an appropriate container and include a cool
    pack (Do not use wet ice. On occasion water from melted ice has
    leaked out of a shipping container causing problems for the mailroom
    staff). Samples must be packed in a leak-proof container (Styrofoam
    box is OK). The lid must be secured with tape and labeled with both
    our address and phone number and your address and phone number

D.  Take your samples to the mail drop for your building by 9:30 am for
    pick-up.

E.  Samples that are not properly packaged (for example-in a zip-loc
    bag, in a mailing envelope, in an interdepartmental mailing
    envelope, taped together, in a 50 ml tube....) will not be accepted
    by the courier.

> For our NIH customers to ship sequencing samples to Tollhouse Ave.:

A.  Call the courier by 10:00 am the day you plan on sending your
    samples. Give them the pick-up location as well as our address.

> B. Follow NIH guidelines for packaging and shipping.
> <http://www.nih.gov/od/ors/ds/shipping/index.html> Package your
> samples in an appropriate container and include a cool pack (Do not
> use wet ice. On occasion water from melted ice has leaked out of a
> shipping container causing problems for the mailroom staff). Samples
> must be packed in a leak-proof container (Styrofoam box is OK). The
> lid must be secured with tape and labeled with both our address and
> phone number and your address and phone number. Your phone number is
> very important. Couriers sometimes have difficulty accessing the NIH
> buildings due to the increased security. They may need to call you to
> let them into the building.

C.  Have your package ready by 11:00 am.

D.  Samples that are not properly packaged (for example-in a zip-loc
    bag, in a mailing

E.  envelope, in an interdepartmental mailing envelope, taped together,
    in a 50 ml tube....) will not be accepted by the courier.

Our shipping address is:

LMT Sequencing Lab

915 Tollhouse Ave.

Suite 211

Frederick, MD 21701

Our phone number is:

301-846-5676

and Fax

301-846-6100

# Labeling-Plates 

> Label the Plate (not the seal, we remove this to process the samples)
> with the [Request Label and Request ID.]{.underline}
>
> If we are performing a G50 purification of your samples, you will need
> to include a plate ID for the primer plate to use for the sequencing
> reaction. If you are sending a primer plate with your request, label
> it with the request label, request ID and [PRIMER]{.underline}.

# Problems

> If any of the above requirements are not met, or if there are any
> questions about your request, you will be notified by the LMT staff.
> Your request will remain on Hold until the problem is resolved and
> this will increase your turnaround time. If the problem can not be
> resolved by e-mail or phone, your samples will be returned to you.

# Special Handling 

If you wish to submit samples in any thing other than standard format,
you

must get pre-appoval from the LMT staff and must include specific

> details along with your samples. Special Handling requests are not
> subject to the standard 3-5 working days.

# Organization and Clutter

> Delete any incorrect or unnecessary requests from the system. Any
> request over five days old for which we have not received samples,
> will be deleted by the LMT staff. If you have need to submit a request
> well in advance of sample shipment please advise the LMT staff so that
> they do not delete your request.