po4 0.2.0

Database of DNA sequences.

PhosphateDB (or PO₄ for short) is a tool for keeping track of DNA constructs such as plasmids, gene fragments, and oligos. It emphasizes keeping track of how each construct was made, and aims to be compatible with existing plasmid-management systems.

The name “PhosphateDB” is based on the fact that DNA molecules are held together by a phosphate backbone, and that the synthesis of new DNA is greatly facilitated by the chemical properties of phosphate. Similarly, this database system aspires to robustly “hold together” DNA sequences in such a way that new sequences can easily be added and shared.

Example

• Your want a tool to better manage your plasmids, but you don’t want to spend a bunch of time re-entering the information for your existing plasmids, which is stored in a big Excel spreadsheet. After installing PO₄, you quickly configure it to recognize the column headers in your spreadsheet. Nothing about your existing spreadsheet needs to change.

• You just designed primers to clone a new plasmid. You enter both the primers and the plasmid into your spreadsheet. While it’s fresh in your head, you also describe how the plasmid will be made in a column labeled “Construction”. The description might look like:

  INV: template=p2 primers=o2,o3

  This syntax indicates that the plasmid is constructed by inverse PCR (“INV”) using the plasmid “p2” as a template and the oligos “o1” and “o2” as primers.

• You just received the primers you designed last week, but you don’t remember exactly how you were planning to use them. You use PO₄—in conjunction with stepwise—to generate an protocol specifying all the relevant details: volumes, concentrations, construct names, etc. If you are making several constructs, the protocol will even group similar constructs and create master mixes of common reagents when possible:

  $ stepwise make p3
  March 27, 2020
  
  $ stepwise make p3
  
  1. Prepare 10x primer mix [1]:
  
     Reagent   Stock    Volume
     ─────────────────────────
     o2       100 µM   0.50 µL
     o3       100 µM   0.50 µL
     water             9.00 µL
     ─────────────────────────
                      10.00 µL
  
  2. Setup 1 PCR reaction and 1 negative control [2]:
  
     Reagent           Stock    Volume
     ─────────────────────────────────
     water                     3.00 µL
     p2             20 pg/µL   1.00 µL
     primer mix          10x   1.00 µL
     Q5 master mix        2x   5.00 µL
     ─────────────────────────────────
                              10.00 µL
  
  3. Run the following thermocycler protocol:
  
     - 98°C for 30s
     - Repeat 35x:
       - 98°C for 10s
       - 60°C for 20s
       - 72°C for 3 min
     - 72°C for 2 min
  
  4. Run 1 ligation reaction:
  
     Reagent              Stock    Volume
     ────────────────────────────────────
     water                        6.75 µL
     T4 ligase buffer       10x   1.00 µL
     T4 PNK             10 U/µL   0.25 µL
     T4 DNA ligase     400 U/µL   0.25 µL
     DpnI               20 U/µL   0.25 µL
     PCR product       50 ng/µL   1.50 µL
     ────────────────────────────────────
                                 10.00 µL
  
     - Incubate at room temperature for 1h.
  
  Notes:
  [1] For resuspending lyophilized primers:
      100 µM = 10 µL/nmol
  
  [2] For diluting template DNA to 20 pg/µL:
      Dilute 1 µL twice into 7*sqrt(DNA) µL

Installation

Install PO₄ using pip:

$ pip install po4

Usage

More details coming soon…