MGH   
CCIB
 
 
CRISPR Sequencing: Sample Submission

 

PLEASE REVIEW OUR GUIDELINES BEFORE PLANNING YOUR EXPERIMENT !

To ensure the successful completion of your CRISPR sequencing project, all guidelines for amplicon design, sample preparation and submission must be addressed. Please contact us if these requirements cannot be met so we can work with you on identifying a solution on a case-by-case basis.

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Amplicon Design:

Amplicons must be 200-280 base pairs in length (including the target-specific PCR primers). The site of interest (i.e. CRISPR cut site) has to be located within the first 100 base pairs (from either the 5'-end or the 3'-end) of the amplicon. Both the NGS adaptors and a unique barcode will be added as part of our NGS sample preparation process.

Our current approach has been designed to generate complete sequence coverage for 200-280 bp amplicons.
If the identification of amplification primer binding sites should present a problem for designing amplicons of this size, the submission of longer amplicons (up to 500 bp) will be acceptable. In this case, however, only the 5'-end and 3'-end of the amplicon will be covered by NGS reads.
To obtain complete sequence coverage of 400-600 bp CRISPR amplicons, please submit your sample(s) for our Complete Amplicon Sequencing service.

IMPORTANT:
Please do not modify your target-specific PCR primers by adding any universal tags!

There is no need to add any universal tags to your amplicons prior to submission. Both the Illumina adaptors and a unique identifier (barcode) will be ligated to your amplicon as part of our NGS sample preparation process. The presence of additional Illumina adaptor sequences will result in complete failure of the sequencing attempt.


Sample Preparation (for individual PCR reactions):

  1. Perform PCR reaction in a total volume of 25 µl or 50 µl.

  2. Analyze a small aliquot of the completed PCR reaction on an Agarose gel to confirm efficient amplification and specific product.

  3. To remove undesired contaminants such as primers, primer dimers, salts and dNTPs, purify the PCR reaction using a PCR column purification kit or a magnetic bead-based cleanup method. Elute the purified amplicons (from column or beads) in ~40 µl of 10 mM Tris-HCl, pH 8.0 or EB Buffer.
    Please note: Our automated workflow requires the submission of 35 µl purified reaction. This allows for a second NGS sample preparation attempt if necessary.

  4. Check quantity of purified amplicon on an analytical gel as no cleanup method will give 100% recovery. Estimate concentration of purified amplicon by comparing it to a reference DNA fragment of similar size and known concentration or determine the DNA concentration using a fluorometric dye assay. We suggest using Invitrogen's Qubit fluorometric assay, which is specific for double-stranded DNA. Quantitation methods like Nanodrop or UV-spec can give inaccurate results as they also measure ssDNA, RNA and other contaminants. The sample concentration should be 10-40 ng/µl.

  5. Submit the purified PCR reaction (total volume: 35 µl) and include a copy of the gel image (of the purified amplicons!) indicating the amplicon size. Please include also a copy of the order form.


Sample Preparation (for 96-well plate orders):

  1. Perform all 96 PCR reactions in the same total volume (25 µl or 50 µl).

  2. Confirm efficient amplification and specific product by Agarose gel electrophoresis.

  3. To remove undesired contaminants such as primers, primer dimers, salts and dNTPs, purify the PCR reactions (you can use a PCR column purification kit or a magnetic bead-based cleanup method). Elute the purified amplicons (from columns or beads) in ~40 µl of 10 mM Tris-HCl, pH 8.0 or EB Buffer.
    Please note: Our automated workflow requires the submission of 35 µl purified reaction. This allows for a second NGS sample preparation attempt if necessary.

  4. Check quantity of purified amplicons on an analytical gel as no cleanup method will give 100% recovery. Estimate concentration of purified amplicons by comparing it to a reference DNA fragment of similar size and known concentration or determine the DNA concentration using a fluorometric dye assay. We suggest using Invitrogen's Qubit fluorometric assay, which is specific for double-stranded DNA. Quantitation methods like Nanodrop or UV-spec can give inaccurate results as they also measure ssDNA, RNA and other contaminants. The average sample concentration should be between 10 ng/µl and 40 ng/µl.

  5. Submit the plate with the purified PCR reactions (with 35 µl in each well) and include a copy of the gel image (of the purified amplicons!) indicating the amplicon size. Please include also a copy of the order form.

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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.

  • We recommend wrapping tubes with Parafilm to prevent samples from drying.

  • 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.

  • If your order contains 48-95 samples or is a full 96 sample plate, please use the plate format to receive a discount (see plate guidelines below).

 

Plate Orders. These guidelines apply to both the 48-95 and full 96 sample plate discounted rates

  • 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.

  • No discounted rate will be applied for orders with fewer than 48 samples, even when submitted in plate format. Multiple orders cannot be combined to receive either the 48-95 or the full 96 plate discount.

  • 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:

We are currently offering our CRISPR Sequencing service for individual samples and full plates once a week, beginning each Wednesday. The deadline for sample arrival at the Core Facility (38 Sidney St. Cambridge) is 4pm. If you should miss this cutoff time, your samples will be entered into the queue and processed during the following cycle.

Samples may be submitted early and they will be stored at the core under the appropriate conditions until the Wednesday start date. Full plates will be processed on a first come, first serve basis and the turnaround time will depend on the current demand. We ask researchers to notify the NGS Team Manager ahead of time to allow us to plan accordingly.

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.