High Volume Submission Policies

All high-volume plate submissions must be submitted in semi-skirted 2mL 96-well plates with clear wells, and sealed with strip caps or sealing film/foil.

Here are some examples of acceptable plate types:

• 96-well Eppendorf twin tec plates Part#951020389,951020346, 951020320, 951020362 (Strip caps Part# 951023035)
• Applied Biosystems Optical 96-well reaction plates Part# N801-0560 (Strip Caps Part# N801-0534
• DOT Scientific PCR plates Part # 351-PCR, 351LP-PCR, 352-PCR, 354-PCR and 650-PCR (Strip Caps Part#405-12PCR)

*The plates are located in the Yale stockrooms or they can be ordered on SciQuest.

**Sealing foil can be purchased from DOT Scientific, Part #T392.

When submitting less than 96 samples on a plate, please submit the samples in consecutive rows. Begin filling the wells at A1 through A12, then B1-B12, C1-12, etc.. If there are any empty wells in the middle of your submission, you will be charged for them.

Cap the plate firmly using the strip caps or sealing film and wrap each plate with parafilm. (Sealing film is preferable to strip caps.)

Be sure to label your submission with a unique name.

High volume plates should be dropped off with a confirmation sheet (available to print after web ordering via our “Sample Submission” page).

Your submission will have the default data output name of [Res Code-Plate Name-Well Name]. If you wish to submit a sample sheet with specific template-primer names for your submission, please fill out the High Volume Sample Sheet and send it to DNAseq@yale.edu.

In addition to our standard protocol, we have alternative sequencing categories you may choose (in our dropdown menu on the ordering page): Difficult Template, shRNA, BAC, single-stranded, and Phage/Lambda. Most of these are self-explanatory; however the Difficult Template protocol might be useful in achieving better sequencing results if your sample falls into any of these categories:

• Is GC-Rich: above 60% GC or concentrated GC content in a small section
• Contains secondary structures
• Has a higher melting temperature/harder to denature
• Has certain di- or tri-nucleotide repeats

All samples must be submitted premixed, with primer and template in one well. The protocols below are sufficient for multiple sequencing reactions with one template/primer combination. This allows us to repeat a sample in case of technical failure without having to contact you for more sample.

DS plasmid DNA:

• 1,000-1,500 ng ds plasmid DNA template
• 2 µl 4 µM primer
• x µl sterile water
• 18 µl total volume

PCR Product:

Template
If 100-300bp:40-50ng
If 300- 500bp:40-160ng
If 500bp- 1kb:500ng

• 2 µl 4 µM primer
• x µl sterile water
• 18 µl total volume

***Nanodrop: Please be sure your initial readings are under 1. Anything over 1 is not accurate and will hinder the sequencing process. If they are over 1, dilute the sample until it is in an accurate range.***

***PCR Cleanup: Please be sure samples are cleaned up prior to submission, not just diluted or desalted. Size exclusion columns are recommended. EtOH precipitation is not recommended as this does not remove unbound dNTPs and primer.***

BAC and Phage Lambda DNA:

• Mix as above except add 4000 ng template DNA.
• Specify BAC or Lambda when ordering.
• Only high purity templates should be used to optimize the amount of DNA loaded onto the sequencer.

Recommended BAC template preparation methods:

1. Alkaline lysis with phenol extraction and isopropanol precipitation.
2. Cesium Chloride Banding

Single-stranded M13 or Phagemid DNA:

• Mix as above except add 200-300 ng template DNA.

Difficult Templates:

• Mix as above
• Specify as Difficult Template when ordering

shRNA:

• Mix as above
• Specify as shRNA when ordering