Jul 26, 2022

Public workspacePreparing 10x PCR buffer

This protocol is a draft, published without a DOI.
  • 1Beneficial Bio, Mboalab
Nadine Mowoh
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Protocol Citation: Nadine Mowoh, Stephane Fadanka 2022. Preparing 10x PCR buffer. protocols.io https://protocols.io/view/preparing-10x-pcr-buffer-cc79szr6
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: Working
We use this protocol and it’s working
Created: July 12, 2022
Last Modified: July 26, 2022
Protocol Integer ID: 66529
Keywords: 10x PCR buffer recipe, Composition of 10x PCR buffer
Abstract
10X ThermoPol Reaction Buffer is optimized for use with Vent® and Deep Vent® DNA Polymerases. This buffer also provides superior reaction conditions for other thermophilic DNA polymerases, includingTaqDNA Polymerase, OpenVent as well as various other DNA and RNA modifying enzymes.

Here we describe the protocol used in making a 10x PCR buffer recipe that provides optimum reaction condition for OpenVent polymerase enzyme.

10x PCR buffer comprise of:

100 mM KCL 100 mM (NH)2SO4 200 mM Tris-HCL pH8.8 @ 25c 20 mM MgSO4 1 % Triton x100
Guidelines
This protocol describes the steps in preparing a low cost 10x PCR buffer.
Materials
Chemicals

ReagentTrisRoche Diagnostics
ReagentHydrochloric acidThermo Scientific
ReagentPotassium chlorideFisher Scientific
ReagentAmmonium sulphateSigma Aldrich
ReagentMagnesium sulphateSigma Aldrich
ReagentTriton X-100Fisher Scientific
ReagentDistilled WaterContributed by users

Materials and Equipment

Sterile Pipette tips (P-10, P200, P-1000)
Micropipettes
Sterile falcon tubes
Sterile Eppendorf tubes

Safety warnings
  • HCL is very corrosive and should be handled with care.
  • Wear protective clothing (Lab coat), face masks and gloves.
Before start
Make sure all chemicals are available in their right concentrations needed to make the 10x PCR buffer.
Preparing reagent stocks
Preparing reagent stocks
Before preparing the 10x PCR buffer, prepare 1M and initial stocks of the different chemicals that would be used.

Note
  • We adopt 3 M stock of HCL to use in preparing Tris-HCL
  • Prepare 10 mL of 3 M HCL by diluting 3.129 mL of concentrated or 37% HCL in 6.871 mL of distilled water.

Safety information
When you mix acid with water, it's extremely important to add the acid to the water rather than the other way around. This is because acid and water react in a vigorous exothermic reaction, releasing heat, sometimes boiling the liquid.


Prepare 10 mL of 1 M concentration of each of the chemical stocks by weighing the salt powders as indicated in the table 1 below:
  • Weigh out the salts as indicated in the table and add to a 100 mL glass beaker.
  • Measure out 10 mL of distilled water and add to the glass beaker.
  • Add a magnetic flea and place on a magnetic stirring plate to mix the solution as shown in figure 1
  • To sterilize, autoclave the solutions by using a steam autoclave at 121° C for at least 30 minutes under at least 15 psi. (All the solutions should be transferred into Duran bottles that can withstand heating).

AB
Chemical Amount needed in (g) for 1 M stock
Tris1.211
KCL0.74
NH(SO4)21.30
MgSO41.20
Table 1


To make 1M Tris HCL pH 8.8

  1. Weigh out Amount1.211 g Tris and add to a 50 mL glass beaker.
  2. Measure out 8 mL of distilled water and add to the glass beaker.
  3. Add a magnetic flea and place on a magnetic stirring plate to mix the solution as shown in figure 1.
  4. Add a pH meter into the solution to observe the pH.
  5. Slowly add the 3 M hydrochloric acid (HCl) solution using a Pasteur pipette to reduce the pH to Ph8.8 . Be careful not to add too much at a time, since the pH will change rapidly.
  6. Once the desired pH has been reached, top up the solution to 10 mL using distilled water.
Figure 1



Critical
Pipetting to make 10x PCR buffer - 1mL
Pipetting to make 10x PCR buffer - 1mL
Pipette the following reagent stocks into a clean 1.5 mL Eppendorf tube as described in the table below to compose 1 mL of 10x PCR buffer.
1M KCL 1M (NH)2SO4 1M Tris-HCL pH8.8 @ 25c 1M MgSO4
Triton x100

AB
Chemical stockAmount required in uL to make a 1000 mL of 10x PCR Buffer
1M KCL100
1M (NH)2SO4100
1M Tris-HCL pH8.8 @ 25c200
1M MgSO420
Triton x10010
Table 2
  • Cork the tube and invert several times to mix the solutions
  • Label the tube appropriately and store at -20c.
Note
  • Before using the buffer, carryout a functionality and Nuclease test to show that the buffer formulation is able to provide a suitable PCR condition for DNA amplification and also free from contaminating nucleases.
  • We typically use our internal quality control protocol to check this.


Pipetting