In this project, our main objective is to check if good quality DNA could be obtained from barley, malt and hop, ingredients utilized in beer brewing. Good quality DNA is essential in DNA fingerprinting approaches that may be employed for detection of ingredients. We have utilized three methods: cetyltrimethylammonium bromide (CTAB) method, QIAGEN DNeasy Plant Mini Kit and Meyer’s method. In order to examine the DNA quality following extraction we implemented three diverse techniques:(i) spectrophotometry to calculate purity utilizing the ratio A260/A280; (ii) agarose gel electrophoresis following DNA extraction to discover the success of the extraction and assess the quantity of high molecular weight DNA and degradation; and (iii) the polymerase chain reaction with four distinct primer pairs, coupled with agarose gel electrophoresis, to find out if the produced DNA can be utilized in downstream applications, see the impact of inhibitors and approximate the fragmentisation of the DNA. The outcomes produced while using previously discussed techniques were then employed to assess the success of each of the extraction methods in their goal of extracting high quality DNA from barley, malt and hop in addition to figuring out whether the treatment of the ingredients influences the DNA quality.
Video: How to extract DNA?
Contents
INTRODUCTION
Table 1 Malt Types
Table 2 Hop Products
MATERIALS AND METHODS
1. DNA EXTRACTION METHODS
1.1 Grinding of samples
1.2 CTAB method
1.3 QIAGEN DNeasy Plant Mini Kit
1.4 Meyer’s method combined with
QIAGEN DNeasy Plant Mini Kit
2. QUALITY ASSESMENT OF DNA
2.1 Spectrophotometry
2.2 Agarose gel electrophoresis
2.2.1 To determine success of DNA extraction
2.2.2 To determine results of PCR primer pair
PLANT 1 and PLANT 2
2.2.3 To determine results of the PCR primer pair rbcL-CRAw Forward and Reverse
2.2.4 To determine results of the PCR primer pair combinations with HOR primers
2.3 Polymerase Chain Reaction (PCR)
2.3.1 PCR with plant-specific primers PLANT 1 and PLANT 2
2.3.2 PCR with plant-specific primers rbcL-CRAw Forward and Reverse
2.3.3 PCR with species-specific primers HOR 1B and HOR 2
2.3.4 PCR with increase of amplicon size using HOR primer pairs
RESULTS
1. DNA EXTRACTION RESULTS
Table 7 Scoring DNA
Series 1: Tobacco leaf and Pilot samples
Table 8 Scoring DNA
Series 2: 9 types of malt and 1 barley variety
Table 9 Scoring DNA
Series 3: 4 types of hop products
2. PCR RESULTS
Table 10 Scoring PCR results
Series 1: Tobacco leaf and Pilot samples
Table 11 Scoring PCR results
Series 2: 9 types of malt and 1 barley variety
Table 12 Scoring PCR results
Series 3: 4 types of hop products
3. SPECTROPHOTOMETRY RESULTS
Table 13 Scoring spectrophotometric results
Series 1: Tobacco leaf and Pilot samples
Table 14 Scoring spectrophotometric results
Series 2: 9 types of malt and 1 barley variety
Table 15 Scoring spectrophotometry results
Series 3: 4 types of hop products
Table 16 Spectrophotometric data…..
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Source: Uppsala University Library