Massachusetts General Hospital Sequencing Core MGH DNA Core Facility Center for Computational and Integrative Biology Directions to the Sequencing Core Sequencing Core - DNA CORE - Molecular Biology - Massachussetts General Hospital
 
 

 

Plasmid Verification & Primer Walking Overview

Sample Preparation


All plasmids being submitted for verification or primer walking must be purified of salts and other contaminants which can negatively affect the quality of data. We ask that you provide us with the following:

  • The maximum size of any plasmid for primer walking cannot exceed 7Kb.
  • The maximum size of any plasmid for plasmid validation cannot exceed 15Kb
  • A text file of the sequence of the plasmid/DNA fragment for plasmid verification
  • For primer walking, provide 100ng of starting primer (conc. 10ng/µL)
  • For primer walking, you must submit a gel picture to verify the size of your plasmid
  • 2µg (10µL at a concentration of 200ng/µL) of template for every 1kb for single strand verification
  • 4µg (20µL at a concentration of 200ng/µL) of template for every 1kb for double strand verification

Plasmid Submission



Under the Sequencing menu, select "Order Plasmid" for either plasmid verification or primer walking. Samples can either be submitted in tube or plate format.

Plasmid Verification

  1. The maximum size of any plasmid for plasmid validation cannot exceed 15Kb
  2. For each sample being submitted, you must enter a Tube ID which may be up to 5 alphanumeric characters long
  3. Under Coverage, select whether you want 1-fold coverage (forward sequencing) or 2-fold coverage (forward and reverse sequencing)
  4. Paste the entire sequence text into the Reference Sequence box. If uploading a file describing your samples, please make sure to review the descriptions of the file formats first
  5. Click on the Submit button to review the information you've provided is correct
  6. Confirm your order and print out the barcoded submission page Please note: For some browsers, the barcode may not print out. As long as the barcode number is present, this will be sufficient for us to process your order.

Primer Walking

  1. The maximum size of any plasmid for primer walking cannot exceed 7Kb.
  2. For primer walking, you must submit a gel picture to verify the size of your plasmid
  3. For each sample being submitted, you must enter a Tube ID which may be up to 5 alphanumeric characters long
  4. Under Coverage, select whether you want 1-fold coverage (1xPrimer Walking, i.e. forward) or 2-fold coverage (2xPrimer Walking, i.e. forward and reverse).
  5. Nothing needs to be entered in the Reference Sequence box
  6. Click on the Submit button to review the information you've provided is correct
  7. Confirm your order and print out the barcoded submission page Please note: For some browsers, the barcode may not print out. As long as the barcode number is present, this will be sufficient for us to process your order.

Sample Drop-Off Points

Samples with properly completed submission forms can be submitted to the Sequencing Core at the following locations:

  • For those who work in the Simches Building: on the 7th floor in the central corridor just outside the doors of the elevator lobby
  • All others on the main MGH campus: Jackson Building, 13th floor, room 1302
  • Charlestown campus: Room 8223, CNY 149 8th floor center, south side
Or you may send your samples to:
    MGH DNA Sequencing Core
    38 Sidney Street
    Suite 100
    Cambridge, MA 02139
For submissions to the Simches Building 7th floor, Jackson 13th floor and the Charlestown Navy Yard please place DNA samples in the Nalgene boxes provided there. In Charlestown the Nalgene boxes must be put inside the cooler in the Sequencing refrigerator or else the samples will not be picked up.

Results


Plasma data will be provided formats:

  1. A compressed .sit file containing the actual sequence data
  2. The consensus sequence in Genbank format
  3. A variance table citing any differences between the sequence provided and the consensus sequence; also in this file will be an alignment between the consensus and reference sequences with variations highlighted in yellow
  4. A file containing the variations of each component sequence as compared to the reference sequence
Note: If submitting samples for primer walking, you will receive only the first two files

Chromatogram files are compressed to facilitate transfer, the files must then be decompressed before viewing. A program that allows viewing and editing of the chromatogram files is also needed. Several commercial programs, in addition to the freeware programs listed below are available to allow viewing and editing of the chromatogram files. Follow the steps outlined below to set up your computer to take advantage of this feature.

  1. Go to Account creation page to set up an account for data retrieval. You must have an account setup prior to sample submission.
  2. Download and install StuffIt Expander (PC) or StuffIt Expander (Mac) on your computer to be used in the uncompression of your data once you retrieve it.. (Follow directions at Aladdin Systems to choose the appropriate version). When you click on the chromatogram file name (*.sit), the file is downloaded from our server and Stuffit Expander saves the file to the desktop or to a location set by you in the Preferences of Stuffit Expander.
  3. Chromatogram Viewers-- 3 freeware programs are listed below. Please install the appropriate viewer on your computer.

Consultations

Consultations with facility personnel are possible but, because of time constraints, should be scheduled separately.

Rates

The rate for plasmid validation and primer walking will be:

  • $17 per kb for 1x Plasmid Validation
  • $22 per kb for 1x Primer Walking
  • 2x double the price
You can choose either single or double stranded verification. Single strand verification is suitable for those who need to verify the sequence of their plasmid. Double stranded verification is suited for those who would like publication quality data.
 
 

  (617) 726-5982