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| - | ==Amplification of Reflectin Genes from the Squid Genomic DNA==
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| - | Two new protocols for genomic DNA extraction were used in order to improve yield and purity of DNA. In addition to three sets of primers allowing for amplification of reflectin, an extra 'positive control' pair of primers was used in the PCR reaction.
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| - | ===DNA Extraction===
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| - | As the [[Team:Cambridge/Experiments/Squid_Dissection_and_Tissue_Sample | previous attempt]] to amplify reflectin genes from ''Loligo vulgaris'' and ''Loligo opalescens'' failed mainly due to low concentration of template DNA, we decided to repeat the experiment, using two, presumably more efficient, new DNA extraction protocols:
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| - | #[http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/mammalian-genomic-dna-miniprep-kit.html Sigma GenElute™ Mammalian Genomic DNA Miniprep Kit] using 'Mammalian Tissue Preparation' procedure
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| - | #[[Team:Cambridge/Protocols/Extraction_of_cleaner_genomic_DNA_from_squid | Improved version of the previously applied zebra fish protocol]]
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| - | Initial steps of the extraction procedure included:
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| - | *Tissue from L.opalescens was homogenized using a pestle and a mortar under liquid nitrogen. In this experiment, we did not extract genomic DNA from the second species, L. vulgaris, in order to save tissue material and reagents.
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| - | *Followingly, a spatula of powdered tissue was transferred to two reaction tubes with different concentrations of Proteinase K:
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| - | :'''GenElute protocol''' - 180 µl of Lysis Solution T and 20 µl of Proteinase K stock solution
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| - | :''final concentration of Proteinase K = 2 mg/ml''
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| - | :'''Zebra fish protocol''' - 198 µl of DNA extraction buffer and 2 µl of Proteinase K stock solution
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| - | :''final concentration of Proteinase K = 200 µl/ml''
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| - | *Samples were kept in a shaking water bath at 50-55°C
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| - | ===Testing the Concentration and Purity of Extracted DNA===
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| - | We measured the DNA content of the supernatant obtained with the GenElute Kit using NanoDrop Spectrophotometer.
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| - | *A260 = 3.89 and using the formula we calculated the concentration of DNA to be
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| - | *However, elevated absorbance at 230nm and a pink colouration of the solution indicates impurities.
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| - | • We decided to conduct PCR using the primers we previously designed. We also added an additional pair of primers that allowed to amplify a mitochondrial gene, thus it served as a positive control.
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| - | ○ Absorbance of 3.89 taken at 260nm. Elevated absorbance at 230nm, and a pink colouration of the solution indicates impurities. (describe somewhere else how you measure DNA content)
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| - | • Template used in all: L.opalsecns genomic DNA A1 tube extracted with DNA Kit from Sigma and different sets of primers:
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| - | ○ A1
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| - | ○ A2
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| - | ○ B1
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| - | ○ Positive control (upload sequences of primers and give reference to a relevant paper) - mitochondrial cytochrome c oxidase subunit I - a pair of primers that allows for amplification of a 710-bp fragment of COI across a broad array of invertebrates
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| - | LCO1490: 5'-ggtcaacaaatcataaagatattgg-3'
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| - | HC02198: 5'-taaacttcagggtgaccaaaaaatca-3
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| - | • Picture of PCR - again no trace of amplified rflectin - no detectable primers and template DNA
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| - | ○ Order of samples 1:A1, 2:A2, 3:B1, 4:+, 5:Ladder IV
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| - | ○ No visible bands in the gel (apart from the Ladder IV)
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| - | ○ Positive control does not work - although in the cited paper, successful use in Loligo pealei is shown
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| - | ○ Suggests that the prepared DNA was not sufficiently clean
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| - | • As we read in some papers (reference), difficult to work with genomic DNA from squids and other cephalopods.
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