Oct 29, 2024
Version 2
Prepare Tris Buffer for Meat identification Protocol V.2
- 1OBS
Protocol Citation: openbioscience Adrian 2024. Prepare Tris Buffer for Meat identification Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg32z1zv25/v2Version created by openbioscience Adrian
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: October 01, 2024
Last Modified: October 29, 2024
Protocol Integer ID: 108970
Abstract
Buffer protocol for meat identification experiment
Materials
reagents: Tris, HCl (12.1M), dd-di-DNAase free water. Note: the dilution of HCL can also be 1M.
devices: scale, thermometer optional: incubator, magnetic stirrer or glass rod for stirring, pipettes, pipette rack, tube rack, pen
labware: 50 ml beaker large enough to permit pH meter measurements
consumables: 50 ml falcon tube
Protocol materials
TrisBio Basic Inc.Catalog #TB0196
In 2 steps
Hydrochloric AcidBioshopCatalog #HCL333.500
Step 3
Before start
Make sure you adjust the pH to the same temperature for the reaction in which the buffer is used.
Prepare Tris Buffer for Meat identification Protocol. Prepare (50 ml) of 0.04 mol/L Tris HCl (pH 7.75)
Prepare Tris Buffer for Meat identification Protocol. Prepare (50 ml) of 0.04 mol/L Tris HCl (pH 7.75)
Add 25 mL of deionized DNase free, distilled-water to a 50 ml beaker.
Measure and add 0.24 g of TrisBio Basic Inc.Catalog #TB0196
Calculation for TrisBio Basic Inc.Catalog #TB0196 weight:
MW for TRIS (C4H11NO3 ) 121.14 mol/Liter
0.04 M TRIS TRIS : m=CMV =0.04mol/L*121.14g/mol *50 ml=0.24 g of TRIS
Add 100 µL HCl 12.1 Molarity (M)
Hydrochloric AcidBioshopCatalog #HCL333.500
Bring the beaker to final experiment desired Celsius temperature (25 °C Celsius for example) and maintain. Can use a water bath or an incubator for example.
Add about 100 µL HCl 12.1 Molarity (M) then iteratively measure pH and add 1 to 2μl HCL until pH reaches 7.75 . The calculation below indicates that about 116uL in total should achieve the desired pH.
!! All this steps are done at the desired temperature set in the previous step. That is required because any pH measurement has to be done at a set temperature as pH is temperature dependent.
Note
The pH indicated in the referenced paper is 7.75 compared to 7 in the main paper.
Note
The initial pH should be high around 10 or 11 as Tris is alkaline and should decrease with the addition of HCl.
Transfer from beaker to a 50 ml falcon tube for storage.
Add water to bring the total to 50 mL .
Store the falcon tube in the fridge for max 4 months.
Protocol references
Normally we make half the solution and titrate with HCl
https://www.researchgate.net/post/How-to-make-a-Tris-HCl-buffer#:~:text=Tris%2DHCl%20can%20be%20prepared,is%20157.6%20g%2Fmol).
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
https://www.reddit.com/r/labrats/comments/193ei6n/trying_to_prepare_trishcl_buffer_ph_seems/?rdt=42146