Introduction

This site contains documents, protocols and suggestions relevant to the procedures performed in the Plasmid Core Facility. The Core provides expert professional services for members of the Cardiovascular Division. Please contact Jon Epstein at epsteinj@mail.med.upenn.edu to inquire further about these services.

» Top

Contact

Jonathan Epstein
954 BRB II/III
421 Curie Blvd
Philadelphia, PA 19104

Email: epsteinj@mail.med.upenn.edu
Phone: 215-898-8731
Fax: 215-573-2094
Lab Phone: 215-898-0252

» Top

Plasmid DNA Service -- How to Submit Your Sample

1. Requesting Plasmid Prep Services -- Download submission form here [doc]
   
   You must submit a complete plasmid map with a description of how the plasmid was created or obtained before the Core will process your request. You will need Filemaker in order to complete the form. You can use Macplasmap to draw a simple picture of your plasmid, or (better) use Macvector to make a sequence file and picture of your plasmid. You must indicate whether any of the plasmid was created by PCR (if so, what portions) and if any of the sequence has been verified by direct sequencing. Also, indicate the steps you took to clone fragments into this plasmid. If the plasmid is to be used for in situs, indicate how it should be linearized and what polymerase should be used for sense and antisense probes.
   
   Email the completed form, with a Map pasted into the appropriate box, and an associated sequence file if you have one, to Rende at rzhang@mail.med.upenn.edu.
   
   For the purpose of keeping good records of every plasmid and for easy retrieval later, the Plasmid Core keeps a glycerol stock and a sample of each DNA preparation. The location of this glycerol stock, a copy of the plasmid map, and other relevant information is stored in the Plasmid Core Database and can be easily searched by MCRC members.
   
   
2. Submitting your E. Coli culture
   
   The Core needs 200 ml of an overnight E. Coli culture for each plasmid. The culture should be prepared the night before, and ready for harvest prior to 9:00 AM on the scheduled day. Label your flask with your name and the name of the plasmid. If necessary, an E. Coli cell pellet can also be used after centrifugation and freezing. This should be approved by Core personnel.
   
   For special requirements, such as low copy number plasmids or large insert sized or BAC clones, please discuss with Core personnel before submission. Special purification procedures may be necessary.
   
   
3. Requesting a Plasmid already in the Core:
   
   You may also request a plasmid that has been previously grown by the Core. Search the Filemaker Pro Core Database and identify the unique Core identification number for the plasmid you want. (This will consist of a Box #, a Row # and a Column letter, e.g. 20-4-G). Then submit a request form and indicate that the plasmid is already in the Core and a new prep is required. The Core will do the rest.

CLICK HERE FOR THE PROTOCOL USED FOR MIDI-PREP PLASMID PURIFICATION.

Expected Results:

1. 100mg to 250 mg plasmid DNA (average) with purity at 260nm/280nm ratio of 1.9 from 200 ml E-Coli culture, with variation based upon the culture conditions, plasmid size and copy number.
2. Plasmid Core will send you one glycerol stock and one tube of purified DNA, while another glycerol stock and DNA sample will be kept by the Core for storage. You can pick up your products in the temporary storage box in 80 degree Core/Epstein lab freezer.
3. DNA purified by this procedure is usually good for gene cloning, cell transfection and homologous recombination. Usage for Transgenic Microinjection is not recommended. DNA for transgenic injection must be prepared using special purification techniques and should be discussed with Core personnel.

 

» Top