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. 2018 Dec 7;13(12):e0203011. doi: 10.1371/journal.pone.0203011

Fig 3. Evaluation of purification of tobacco genomic DNA and total RNA using filter paper-based spin columns with respective Qiagen kit buffers and homemade buffers.

Fig 3

(A) Agarose gel electrophoresis for 2.5 μl tobacco genomic DNAs elution from purification experiments using Qiagen DNeasy plant mini kit buffers with Qiagen original spin column (Lane Q/Q), filter paper recharged used spin column (Lane Q/R) and filter paper-based homemade spin column (Lane Q/H*), followed by tobacco genomic DNAs purified using homemade buffer with Qiagen original spin column (Lane H/Q), filter paper recharged used spin column (Lane H/R) and filter paper-based homemade spin column (Lane H/H*). (B) UV spectrum curve of tobacco DNAs purified using Qiagen kit (Q/Q, black curve), filter paper recharged spin columns with Qiagen kit buffers (Q/R, blue curve) or homemade buffers (H/R, red curve) from the same amount leaf tissue. Y-axis is UV absorbance, and X-axis is wavelength (nM). (C) Amplification plots for three duplicated qPCR reactions contain 20 ng DNA purified using Qiagen kit (Q/Q, Blue curves) or DNA purified from filter paper recharged spin column with homemade buffer (H/R, Red curves) respectively. The x-axis is PCR cycle numbers, Y-axis is the level of SYBR fluorescence, and the green line is an arbitrary threshold to determine the Cq value (the fractional cycle number at which amplification curve meet threshold level). (D) MOPS-formaldehyde denaturing agarose gel electrophoresis separated 5 μl RNA purified using Qiagen RNeasy plant mini kit buffers with a Qiagen original spin column (Lane Q/Q), filter paper recharged used spin column (Lane Q/R) and homemade filter paper-based spin column (Lane Q/H*), followed total tobacco RNAs purified by using homemade buffer with Qiagen original spin column (Lane H/Q), filter paper recharged used spin column (Lane H/R) and filter paper-based homemade spin column (Lane H/H*). (E) UV spectrum of tobacco total RNA purified using Qiagen kit (Q/Q, black curve), filter paper recharged spin column with Qiagen RNeasy plant mini kit buffers (Q/R, blue curve) or homemade buffers (H/R, red curve). Y-axis is UV absorbance, and the X-axis is wavelength. (F) Amplification plots of three duplicated qRT-PCR reactions for 2.5 ng RNA purified using Qiagen kit (Q/Q, Blue curves) or RNA purified using filter paper recharged spin column with homemade buffer (H/R, Red curves) respectively. Note: * The starting material amount is 100 mg tobacco leaf tissue for experiments using a Qiagen spin column or filter paper recharged spin column, and half amount of plant sample (50 mg) used for homemade spin column purification. All DNAs or RNAs were eluted using 100 ul elution solution.