===========================
DNase (2X) and papain (2X)
===========================

DNase and papain from 2X
=============================

This is based off of `Worthington Papain Dissociation System <https://www.worthington-biochem.com/products/papain-dissociation-system>`_ to achieve a final ** 20 U papain/mL + 2,000 U DNase I/mL solution with 0.5mM EDTA and 1mM L-cysteine**. 

Add equal volumes 2X DNase and papain from the -80°C. e.g. 1 mL DNase (2X) + 1 mL papain (2x).

================================   ========================================================
 **Solution**                       **Details**
================================   ========================================================
 :ref:`DNase (2X) <DNase2X>`         4,000 U/mL in DMEM/F12
 :ref:`Papain (2X) <Papain2X>`       40 U/mL with 1mM EDTA, 2mM L-cysteine in DMEM/F12
================================   ========================================================

.. note:
  This solution can be stored at -20°C for a few weeks (not thoroughly tested).

.. _Lcys_for_papain:

L-Cysteine (50 mM)
======================
1. Measure 60.6 mg L-Cysteine (MW: 121.16 g/mol) and add to a 15 mL conical
2. Add 10 mL DMEM/F12
3. Take into TC and sterilize by filtering through a 0.22 µm filter
4. Aliquot ~1.1 mL into 1.6 mL eppendorf tubes and freeze with papain aliquots at -80°C

.. note:
  You can measure the weight of the 15 mL conical before and after adding the L-Cysteine to account for the weight loss when transferring this small amount. If done well however, it should be fairly negligible. Adjust DMEM/F12 as necessary.


.. _Papain2X:

Papain (2X: 1mM EDTA, 2mM L-cysteine, 40 U papain/mL) 
==================================================================

.. note:
  We add all the L-cysteine and EDTA into the 2X papain (DNase will only be dissolved in DMEM/F12) so we make a stock 2X papain solution that is ** 1mM EDTA, 2mM L-cysteine, 40 U papain/mL ** to achieve a final concentration of 0.5mM EDTA, 1mM L-cysteine, 20 U papain/mL.

To make a 50 mL solution of 2X papain from `Papain 100 mg <https://www.fishersci.com/shop/products/papain-i-carica-papaya-i-latex-2/AAJ61875MC>`_:

=============================== ================ ===============
 Component                       Final conc       Volume
=============================== ================ ===============
EDTA (0.5M, pH=8.0)               1 mM             100 µL
L-Cysteine (50mM)                 2 mM             2 mL
Papain (100 mg bottle)            40 U/mL          100 µL
DMEM/F12                                           47.9 mL
=============================== ================ ===============

Sterilize with 0.22 µM filter and store aliquots at -80°C. :ref:`EDTA (0.5M, pH=8.0) <EDTA_for_papain>` should be in Bruni and :ref:`L-Cystein (50mM) <Lcys_for_papain>` should be in -80°C with papain aliquots.


.. _DNase2X:

DNase (2X: 4,000 U/mL solution)
==================================

To make a 2X solution from `DNase I (100 mg, 2000 units/mg) <hhttps://www.stemcell.com/products/dnase-i.html>`_.

Add 50 mL DMEM/F12 to a 100 mg bottle, sterilize with 0.22 µM filter and store aliquots at -80°C.