Add 1/6 volume of this DNA Loading buffer to your DNA sample, for example 5 µL DNA sample and 1 µL buffer.

Often we have more concentrated DNA solutions as for example a normal DNA miniprep of a plasmid where about 2 µL DNA sample is enough for checking size and integrity. If you have many samples, mix one volume (for example 10 µL ) 6 x DNA loading buffer with two volumes of water (for example 20 µL ). Mix well and put 4 µL drops of the mixture on a piece of Parafilm and mix each drop with 2 µL of DNA sample. This mixture will have the right fraction of loading buffer.

The table below contain the components of a 6x loading buffer used in the lab. It has two colors one bluish that migrates slowly and one yellow that migrates rapidly. This loading buffer should be compatible with PCR (see below). Note that the buffer should contain either Orange G or Tartrazine as the yellow color. Orange G is preferred since it migrates faster.

Recipe for 50 mL 6x DNA loading buffer:

• add 6.25g FICOLL 400 powder to a new 50 mL FALCON tube.
• add ddH2O to about 30 mL, shake to dissolve. This may take time, heating the tube in a water bath helps.
• add 7.5 mg Xylene Cyanol FF powder
• add 7.2 mL Orange G (1% w/v) OR 900 µL of VAHINE yellow contains Tartrazine
• add water to 50 mL

The resulting solution is about the same colour as the GoTaq green flexi buffer commercialized by Promega. The gel below shows GoTaq green flexi buffer loaded on gel.

This loading buffer should be compatible with PCR (see below), add it to 16% (v/v) of the final PCR volume or it can be added to 32% (v/v) of a 2x PCR mastermix.

The yellow food coloring contain Tartrazine (E102). In this case, exact concentration is not known, but could be measured using a spectrophotometer at the absorption maximum of 472 nm. The molar absorptivity of Tartrazine at 472 nm is 20,330 L mol-1 cm-1.

From invitrogens website I found the following information:

Xylene Cyanol (0.02%), Tartrazine and Ficoll (2.5%) do not inhibit this enzyme (recombinant Taq).

The information was avaliable thorugh this link.

I could not find information for PHUSION, but I assume this enzyme is not more sensitive.

This suggests that 2.5% ficoll could be added to a PCR reaction.

Kramer 2001 suggests to add 0.5 - 1% Ficoll 400. Surpisingly they refer to v/v:

add Ficoll 400 to a final concentration of 0.5% to 1% (v/v)

Kramer, M. F., & Coen, D. M. (2001). Enzymatic amplification of DNA by PCR: standard procedures and optimization. Current Protocols in Molecular Biology / Edited by Frederick M. Ausubel... [et Al.], Chapter 15, Unit 15.1.

PCR mastermix can tolerate 0.0025% Xylene Cyanol FF in the final solution. For example, commercial https://international.neb.com/products/m0271-quick-load-taq-2x-master-mix#Product Information yields 0.0025% XyleneCyanol FF in the final 1x PCR mixture:

!

The final loading buffer should have 0.0025% (w/v) dye. The loading buffer is 6x concentrated, so the loading buffer should have 0.0025 * 6 = 0.015% (w/v). If we assume a density of 1 g/mL (the final buffer is denser), we should add (0.015/100) * 50000 = 7.5 mg of dye to 50 mL.

Commercial 2X Quick-Load Taq Master Mix has 0.024% Orange G (see above).

Hoppe 1992 tests various dyes for their compatibility with PCR. This paper is old and I have not been able to find any version of it on-line. However, a more recent paper refer to it, Prigge 2001 and they make the following claim:

To facilitate higher throughput PCR mapping, loading dyes and sucrose were included in the reaction mixtures (Hoppe et al., 1992). PCR reactions contained 50 mM KCl, 10 Tris-HCl, pH 8.3, 1 mM MgCl2, 0.1 g/L BSA, 12% sucrose, 0.1% Triton X-100, 0.2 mM cresol red, 0.3% yellow food coloring (McCormick and Co., Inc., Hunt Valley, MD), 125 M each dNTP, 250 nM each primer, and 0.1 U/ L Taq polymerase.

The McCormick food coloring seem equivalent to the VAHINE available in Portugal (see above). In our recipe the final concentration is (0.9 mL / 50 mL ) / 6 = 0.3 % (v/v).

VAHINE food coloring comes in a package with blue, red and yellow. It has the following components:

• http://www.wendychao.com/science/protocols/loading_buffer/
• http://www.protocol-online.org/biology-forums-2/posts/12397.html
• http://www.geneticorigins.org/pv92/recipes2.htm
• https://www.researchgate.net/post/Which_dye_doesnt_inhibit_PCR_reaction