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The Applied Genomic Technologies Core

Sample Submission Guidelines

In order for automated DNA sequencing to be successful, two factors are crucial: (1) the template must be of high purity and (2) the template must be accurately quantified. Failure to meet either criterion may result in poor or no useful sequence data.

Each sample tube should consist of the Template DNA AND Primer combined in a 0.2 ml 8-strip PCR tube in a final volume of 21µl. 96-well plates permitted for orders between 48-95 samples. Follow the instructions below for required DNA amounts based on the Template type.

Universal Primers and Strip Tubes/Caps are available by request.

For Strip Tube/Cap requests, please specify a quantity of 96, 240 or 480 reaction tubes/caps. Requests should be emailed to: gcfac@health.state.ny.us.

Universal Primers

100 reaction aliquots for general use.
500 reaction aliquots for high throughput users.

The Core has aliquots of universal primers available for pickup in the freezer in room 1054 at DAI. For those at CMS, stop by room 4102 and ask a sequencing staff member.

For those at ESP or Griffin Lab – To request primers, send an email to: gcfac@health.state.ny.us. Please specify primer name and aliquot size.
Please allow 2 days to receive requested items.

Required consumables:

• 0.2 ml PCR strip tubes (Axygen; Cat# PCR-0208C) Krackeler #383-PCR-0208C-PK (for 125 strips of 8 tubes)
• 0.2 ml PCR strip caps (Axygen; Cat# PCR-02CPC) Krackeler #383-PCR-02CPC-PK (for 125 strips of 8 caps)
• 96-well PCR plates - see instructions below

PCR Products

1. PCR products must be supplied in an amount determined by the fragment length. This is equal to the fragment length multiplied by 0.1. For example, a 500 bp PCR product sample should contain (0.1)(500) = 50 ng total DNA.
2. 10 pmol of Primer, whether universal or unique.
3. Additional ddH2O, up to final volume of 21µl

Plasmids

1. 1ug of Template.
2. 10 pmol of Primer, whether universal or unique.
3. Additional ddH2O, to final volume of 21µl

Cosmids

1. 2ug of Template.
2. 10 pmol of Primer, whether universal or unique.
3. Additional ddH2O, to final volume of 21µl

BACs *Note: BACs ONLY - Template and Primers in separate 1.5ml tubes.

1. Template amount - 250ng/µl, 2ug minimum per reaction.
2. Primer - 3.2pmol/µl, minimum 20 pmol per reaction.

Entering a Submission Request in the dnaLIMS system:

Instructions for using the dnaLIMS system can be found at:
http://www.wadsworth.org/cores/applied_genomics/sequencing/instructions.htm.

Login to dnaLIMS: http://corelims.wadsworth.org Each person should have their own account with up-to-date contact information. Labs should not use shared logins

Select "Enter Sequencing Requests"

Step 1. Fill in the number of reactions to be submitted.

Step 2. Select the appropriate Service Type; A, B, or C below:

1. PCR
2. Clinical PCR - Clinical Use Only -
   DO NOT SEND INFECTIOUS OR CONTAMINATED SAMPLES OR PACKAGING.
   
   This Service Request must be used for all clinical specimens. Use your laboratory accessioning number as your template name on this form. Be advised that the requesting laboratory is responsible for the interpretation of any sequence data generated as well as the archival of all appropriate materials in accordance with CLEP regulations. If you require emergency prioritization, your request must be accompanied by a brief letter from your Divisional Director confirming your needs. Failure to follow these instructions will result in the rejection of your sample.
3. Plasmid, Cosmid, or BAC

Step 3. Fill out information completely

• Project Code/Fiscal Account # (SP/LR): When you submit an order through the LIMS system, please enter the 10 digit project code.
• HRI #: In boxes where you are asked for the HRI number, enter the 8-digit HRI number.

NOTE: Prior written approval from Division Director required for use of State funds for chargebacks. If State funds are to be used instead of a grant, enter xx-xxxx-SP or xx-xxxx-LR in the space provided for the HRI # to indicate whether to charge the State Purpose or Laboratory Reference account for that project code. Ask your principal investigator for correct account information.

• Template/Primer information - Sample name, sample concentration, fragment size, primer name.
• Enter any comments such as high AT or GT content, G-C rich or request for dGTP chemistry in the comments section.

Please check Sample/Primer information carefully for inaccuracies before clicking the "Submit" button.

Select the "Submit" button.

Step 4. Print two (2) copies of the order request

• Submit one authorized and dated copy with your samples.
• Keep the second copy for your records.

Strip Tube Labeling Instructions:

Strip tubes should be labeled by writing the order number (as received through dnaLIMS) down the side of the first sample tube using an alcohol-resistant Lab Marker. Sharpies DO NOT work. Test marker to make sure it does not rub off. Then label each tube 1 thru 8. The first tube in each strip of 8 should again be labeled with the order number. An example of labeling of a 12-sample submission is shown below.

Samples should be placed in a sealed plastic bag with the order number written on it. The zip-lock bag and the signed order are to be placed in an envelope addressed to:

Applied Genomic Technologies Core (AGT Core)
DAI -20C freezer room 1054
Service: DNA Sequencing
From: your name and phone number

The envelope may be sent through inter-agency mail, shuttle-driver, brought in person to the -20ºC freezer in Room 1054 DAI, or placed in the sequencing drop-off box in the cold room at 4107 CMS.

96-well PCR Plate Submission Instructions:

• Minimum of 48 samples
• Maximum of 95 samples. Leave H12 empty for Core control sample.
• Plates must be well sealed; strip caps recommended.
• Label plate with order #, user name
• Wells must be filled A1-H1, A2-H2, A3-H3, etc. in the same order the reactions are listed on the order form: A1 -1st sample, B1- 2nd sample, etc.
• Do NOT skip any wells!
• Same quantity DNA, Primer, Final Volume as 8-strip tubes.

Samples should be placed in a sealed plastic bag with the order number written on it. The zip-lock bag and the signed order are to be placed in an envelope addressed to:

Applied Genomic Technologies Core (AGT Core)
DAI -20C freezer room 1054
Service: DNA Sequencing
From: your name and phone number

The envelope may be sent through inter-agency mail, shuttle-driver, brought in person to the -20ºC freezer in Room 1054 DAI, or placed in the sequencing drop-off box in the cold room at 4107 CMS.

AGT Core-supplied Universal Primers

Universal Primers are available in:

100 reaction aliquots for general use.
500 reaction aliquots for high throughput users.

The Core has aliquots of universal primers available for pickup in the freezer in room 1054 at DAI. For those at CMS, stop by room 4102 and ask a sequencing staff member.

For those at ESP or Griffin Lab – To request primers, send an email to: gcfac@health.state.ny.us. Please specify primer name and aliquot size.
Please allow 2 days to receive requested items.

Primer	Sequence (5'-3')	Melting Temp (ºC)
M13-20:	GTA-AAA-CGA-CGG-CCA-GT	52
M13REV:	AAC-AGC-TAT-GAC-CAT-G	46
*T3HT:	AAT-TAA-CCC-TCA-CTA-AAG-GG	49
T7P:	AAT-ACG-ACT-CAC-TAT-AGG-G	48
T7term:	GCT-AGT-TAT-TGC-TCA-GCG-G	53
SP6:	GAT-TTA-GGT-GAC-ACT-ATA	44
SK:	TCT-AGA-ACT-AGT-GGA-TC	48
KS:	CGA-GGT-CGA-CGG-TAT-CG	56

*This high temperature (HT) version matches many common vectors and are recommended for cycle sequencing. Be sure to check the sequence against your vector, as this primer differs from the commonly used T3 primers.

T3HT (5’-3’) AAT-TAA-CCC-TCA-CTA-AAG-GG replaces

T3 (5’-3’) - ATT-AAC-CCT-CAC-TAA-AG.

Questions: contact Matt Shudt, 474-4200, shudt@wadsworth.org