TITLE: Meat identification protocol
AUTHORS: openbioscience Adrian

[DESCRIPTION]
Identifying species by identifying unique DNA sequences is common practice. 
In this case we will identify the presence or absence of pork DNA in a meat sample.
We will use the alkaline method for DNA extraction and LAMP method for DNA amplification and detection.
This is not a new protocol but rather the detailing of a previously published protocol listed in the manuscript citation.

[BEFORE_START]
Pork meat identification protocol. Meat Preparation protocol based on
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3550954/
"About 500 mg of meat was triturated in pestle & mortar with four volumes (4 ml) of 0.2N NaOH. 5 μl of the extract was again mixed with eight volumes (40 μl) of 0.2N NaOH in a sterile micro centrifuge tube. Mix was heated at 75 °C in water bath for 20 min and then added eight volumes (360 μl) of 0.04 M Tris HCl (pH 7.75) for neutralization of pH. 1 μl of final mix containing about 100 ng DNA was used for."

[GUIDELINES]
Follow the country specific guidelines.

[STEPS]
SECTION: Meat Preparation
1. Measure 500 mg pork meat without fat. This is our sample (aliquot.
3. Measure 1 ml of the liquid meat and add it to a sterile centrifuge tube labeled "raw sample". This is done with a 100- 1000 μl micropipette.
4. Prepare 5 ml of NaOH 0.2  mol/L (sodium hydroxide or caustic soda). This will be used twice, once 4 mL and once 40μL.
5. Add 4 ml from the  tube labelled "NaOH 0.2 mol/L" to the "raw sample" tube. This will break down the cell membranes freeing the DNA and also break down (denaturate) some of the proteins.
6. Measure 5 μL from previous step and add it to a new sterile centrifuge tube labeled "prepared sample". This is done with a 1- 10 μl micropipette.
7. Add in the "prepared sample" new tube 40μL from the  tube labelled "NaOH 0.2 mol/L".
8. Heat the resulting mix at 75 Celsius for 20 minutes in a water bath or using the LAMP device or a PCR device. This will further break down the cell membranes freeing the DNA.
9. Prepare (360 μl) of 0.04 mol/L Tris HCl (pH 7.75).
This will be done by mixing Tris with HCl

MW for TRIS Hydrochloride (C4H11NO3 ·HCl) 157.6 mol/Liter



0.04 M TRIS TRIS Hydrochloride: m=CMV =0.04mol/L*157.6g/mol *360uL=2.26 mg of TRIS Hydrochloride

Normally we make half the solution and titrate with HCl 
https://www.reddit.com/r/labrats/comments/193ei6n/trying_to_prepare_trishcl_buffer_ph_seems/?rdt=42146

https://www.cshlpress.com/pdf/sample/2014/LabRefV1/LabRefV1Ch1S1.pdf has collection of reagents
pH calculations= https://www.youtube.com/watch?v=387t-nh0OMg
https://www.cusabio.com/m-296.html#a21 for calculating Tris HCL pH from Tris and Hydrocloric acid

4.1. In a new centrifuge tube labelled "NaOH 0.2  mol/L" add 5 mL DNAfree water and 40 mg or NaOH. Mix to dissolve manually or using a vortex.

For general molarity calculations see Molarity calcualation
Molar concentration is the number of moles contained in one Liter of a substance. The concentration of a solution is expressed in mol/L.	
C=(m/M)/V where  V is the volume in liters m is mass in grams and M is molar weight? in grams/mol
https://www.alloprof.qc.ca/en/students/vl/sciences/calculating-molar-concentration-s1622
----
Molecular Weight of sodium hydroxide (NaOH) =40 g/mol: Na=22.989g/mol O=15.999g/mol H=1.008g/mol (http://chembook.org/page-nonav.php?chnum=1§=5)   so: 22.989g/mol+15.999g/mol+1.008g/mol=39.996g/mol.
 
so for us: m=C*M*V =0.2mol/L*40g/mol *5mL= 8 g/L*5 mL=40 mg of NaOH

10. Add 360 μl of the 0.04 mol/L Tris HCl. This is done for for neutralization of pH.
11. Sample 1 μl in a PCR tube and store at -20.
SECTION: LAMP
12. 
SECTION: Meat Preparation
2. Grind (triturate) the meat in a pestle and mortar. This will break the structure and results in a paste/liquid.