Funding for this research was provided in part by the USDA-ARS Research Associate Program and the USDA Crop Germplasm Committee.

1. Amylose Determination from Potato Tuber Starch
<ol>
	<li>Peel and cut fresh tubers into small cubes.</li>
	<li>Place potato cubes in small brown bags and store overnight at -80 &#176;C.</li>
	<li>Transfer potato cubes to nylon bags and for freeze drying.</li>
	<li>After freeze dried, grind the potato sample into powder using mortar and pestle or a Wiley Mill.</li>
	<li>In a 50 ml tube, add 20-30 mg of the freeze-dried, ground tuber sample.</li>
	<li>Prepare a solution of 45% (w/v) perchloric acid (mix 24.4 ml of 60% perchloric acid and 25.6 ml of ultra-pure water.</li>
	<li>Add 500 &#956;l of 45% (w/v) perchloric acid and shake or swirl it to disperse starch granules.</li>
	<li>After a four-minute incubation period at room temperature, add 16 ml of ultra-pure water to the solution and mix by vortexing.</li>
	<li>After non-soluble material has settled to the bottom of the tube (7-10 min&#160;later), transfer 40 &#956;l of the solution to a microtiter plate (avoiding the pipetting of any particles).</li>
	<li>Add 50 &#956;l of iodine solution (2 g KI + 1 g I2 in 900 ml of ultra-pure water) and mix the sample by pipetting.</li>
	<li>Read the absorbance at 550 nm and 620 nm immediately.</li>
	<li>Determine the amylose percentage after comparing the amylose/amylopectin ratio of each sample (620 nm/550 nm absorbance) with a standard curve generated from amylose and amylopectin solutions at a range of concentrations. This procedure will be described next. Read the blank (iodine solution and perchloric acid) along with the test samples. Use blanked data for the analyses.</li>
</ol>
2. Amylose/amylopectin Curve
<ol>
	<li>In separate tubes weigh 12.5 mg of amylose and 12.5 mg of amylopectin.</li>
	<li>Add to each tube 5 ml of 45% (w/v) perchloric acid and dissolve completely.</li>
	<li>For each of the solutions (amylose and amylopectin), bring to a final volume of 50 ml with ultra-pure water.</li>
	<li>Mix 6.25 ml of amylose stock from the previous step with 18.75 ml ultra-pure water. This will make a 6.25 mg/ml amylose solution.</li>
	<li>Repeat the procedure for the amylopectin solution.</li>
	<li>Using the amylose and amylopectin standard solutions from steps 2.4 and 2.5, prepare the percent amylose standards which are 0-100% amylose (100-0% amylopectin) to a final volume of 5 ml to create the standard curve in 10% intervals. 
	For example, to prepare: 
	The 0% amylose / 100% amylopectin standard, pipette 5 ml amylopectin solution. 
	The 10% amylose / 90% amylopectin standard, combine 0.5 ml amylose and 4.5 ml amylopectin solutions. 
	The 20% amylose / 80% amylopectin standard, combine 1.0 ml amylose and 4.0 ml amylopectin solutions. 
	The 30% amylose / 70% amylopectin standard, combine 1.5 ml amylose and 3.5 ml amylopectin solutions. 
	The 40% amylose / 60% amylopectin standard, combine 2.0 ml amylose and 2.0 ml amylopectin solutions. 
	The 50% amylose / 50% amylopectin standard, combine 2.5 ml amylose and 2.5 ml amylopectin solutions. 
	The 60% amylose / 40% amylopectin standard, combine 3.0 ml amylose and 2.0 ml amylopectin, and so on).</li>
	<li>Transfer 40 &#956;l of each standard mixture to a microtiter plate. Include a well with 40 &#956;l 45% (w/v) perchloric acid as the blank.</li>
	<li>Add 50 &#956;l of iodine solution (2 g KI + 1 g I2&#160;in 900 ml of ultra-pure water) and mix each sample (including the blank) by pipetting.</li>
	<li>Read absorbances at 550 nm and 620 nm immediately and calculate the amylose/ amylopectin ratio for each concentration.</li>
</ol>

<ol>
	<li>Composition Hoover, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=molecular+structure,+and+physicochemical+properties+of+tuber+and+root+starches:+a+review.">molecular structure, and physicochemical properties of tuber and root starches: a review.</a> Carbohydr. 45 (3), 253-267 (2001).</li><li>Bertoft, E., Blennow, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Potato+Starch.">Structure of Potato Starch.</a> Advances in Potato Chemistry and Technology. , 83-98 (2009).</li><li>Campbell, M. R., Yeager, H., Abdubek, N., Pollak, L., Glover, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+methods+for+amylose+screening+in+maize+starches+from+exotic+backgrounds.">Comparison of methods for amylose screening in maize starches from exotic backgrounds.</a> Cereal Chem. 79, 317-321 (2002).</li><li>Zhu, T., Jackson, D. S., Wehling, R. L., Geera, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+amylose+determination+methods+and+the+development+of+a+dual+wavelength+iodine+binding+technique.">Comparison of amylose determination methods and the development of a dual wavelength iodine binding technique.</a> Cereal Chem. 85, 51-58 (2008).</li><li>Hovenkamp-Hermelink, J., Devries, J., Adamse, P., Jacobsen, E., Witholt, B., Feenstra, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+estimation+of+the+amylose+amylopectin+ratio+in+small+amounts+of+tuber+and+leaf+tissue+of+the+potato.">Rapid estimation of the amylose amylopectin ratio in small amounts of tuber and leaf tissue of the potato.</a> Potato Res. 31, 241-246 (1988).</li><li>Shannon, J. C., Garwood, D. L., Boyer, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+Physiology+of+Starch+Development.">Genetics and Physiology of Starch Development.</a> Starch. , 23-82 (2009).</li><li>Haase, N. U., Plate, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+potato+starch+in+relation+to+varieties+and+environmental+factors.">Properties of potato starch in relation to varieties and environmental factors.</a> St&#228;rke. 48 (5), 167-171 (1996).</li><li>Johnston, F. B., Urbas, B., Khanzada, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+storage+on+the+size+distribution+and+amylose/amylopectin+ratio+in+potato+starch+granules.">Effect of storage on the size distribution and amylose/amylopectin ratio in potato starch granules.</a> Am. Pot. J. 45, 315-321 (1968).</li><li>Gibson, T. S., Solah, V. A., McCleary, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+procedure+to+measure+amylose+in+cereal+starches+and+flours+with+concanavalin+A.">A procedure to measure amylose in cereal starches and flours with concanavalin A.</a> J. Cereal Sci. 25 (2), 111-119 (1997).</li><li>Sun, S., Matheson, N., Yun, S. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+amylose+content+of+starches+after+precipitation+of+amylopectin+by+concanavalin+A.">Estimation of amylose content of starches after precipitation of amylopectin by concanavalin A.</a> Starch. 42, 302-305 (1990).</li></ol>

We have nothing to disclose.

Amylose content in potato tubers typically ranges from 20-35%7,8. If most values are outside this range, consider these possible errors: 1) raw data were used instead of blanked data for the construction of the amylose/amylopectin standard curve; 2) raw data were used instead of blanked data for the determination of the amylose/amylopectin ratios from the potato samples; or 3) the water used for the analysis was not pure enough. We noticed that by using distilled water and not ultra-pure water in the analysis,...

Approximately 80% of the fresh weight of a potato tuber is water; nearly all of the remaining dry matter is starch1. Most of the starch (70%) is composed of amylopectin, while the remainder is amylose. The ratio between amylose and amylopectin is the most important property influencing the physical properties of starch. Amylose is a linear alpha 1-4 glucose chain, while amylopectin is a linear alpha 1-4 chain with alpha 1-6 branches2. Methods such as iodine binding, differential scanning calorimetry (DSC), high performance size exclusion chromatography (HPSEC) and concanavalin A interactions have been developed for starch determination in different crop species3,4. Each protocol requires special skills and equipment, making it difficult to scale up to quantify numerous samples at a time. The method we present here is a modified protocol from Hovenkamp-Hermelink et al.5 which is a dual wavelength iodine binding method based on stained starch granules. The advantages of this method versus others include the amylose determination from raw starch without purifying it and the use of the dual wavelength system to increase the precision of the method4,6.&#160;

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td> Reagents</td>
 </tr>
 <tr>
 <td>Amylose </td>
 <td>Sigma</td>
 <td>859656</td>
 <td></td>
 </tr>
 <tr>
 <td>Amylopectin </td>
 <td>Sigma</td>
 <td>A8515</td>
 <td></td>
 </tr>
 <tr>
 <td>Perchloric Acid 60%</td>
 <td>Sigma</td>
 <td>311413</td>
 <td>Very hazardous to skin and eyes</td>
 </tr>
 <tr>
 <td>Iodine</td>
 <td>Sigma</td>
 <td>23214TD</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium iodide</td>
 <td>Sigma</td>
 <td>08625JE</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Plate Reader</td>
 <td>Bio Tek</td>
 <td>ELx800</td>
 <td></td>
 </tr>
 </tbody>
</table>

rapid high throughput amylose determination in freeze dried potato tuber samples

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones.&#160;

The standard curve constructed from blanked data of different amylose/amylopectin concentration solutions shows approximate absorbance ratios ranging from 0.7 to 1.6 (Table 1). A linear regression trend line from these data is later used for inferring amylose content in freeze dried potato samples (Figure 1).
The plant production environment has some effect on the amylose content in the potato tuber7. Replicated amylose content determinations in two...

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Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in ...

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13.9K Views.  University of Wisconsin - Madison. This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance va...

Video: Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

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A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in ...

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

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New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

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The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample preparation is a key and often complex procedure, particularly for biological samples. Although X-ray speciation techniques are widely used, no detailed protocol has been yet disseminated for users of the technique. Further, chemical state modification is of concern, and cryo-based techniques are recommended to analyze the biological samples in their near-native hydrated state to provide the maximum preservation of chemical integrity of the cells or tissues. Here, we propose a cellular preparation protocol based on cryo-preserved samples. It is demonstrated in a high energy resolution fluorescence detected X-ray absorption spectroscopy study of selenium in cancer cells and a study of iron in phytoplankton. This protocol can be used with other biological samples and other X-ray techniques that can be damaged by irradiation.

This protocol presents a detailed procedure to prepare biological cryosamples for synchrotron-based X-ray absorption spectroscopy experiments. We describe all the steps required to optimize sample preparation and cryopreservation with examples of the protocol with cancer and phytoplankton cells. This method provides a universal standard of sample cryo-preparation.

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample ...

Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples

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Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

Extraction and Purification of Polyphenols from Freeze-dried Berry Powder for the Treatment of Vascular Smooth Muscle Cells In Vitro

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution X-Ray Absorption Spectroscopy