Package: po4
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 ofhoweach 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.ExampleYour 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,o3This 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 withstepwise—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) µLInstallationInstall PO₄ usingpip:$ pip install po4UsageMore details coming soon…