Amplification of Reflectin Genes from the Squid Genomic DNA

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.

DNA Extraction

As the 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 new DNA extraction protocols:

1. [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]

• We used two new, presumably more effective, DNA extraction protocols. ○ Sigma GenElute Mamalian Genomic DNA extraction Kit, using 'tissue protocol' ○ Improved zebrafish protocol § Homogenize tissue from L.opalescens using pestle and mortar under liquid nitrogen (is L.opalsecens the only species we worked on?) § Transfered a spatula fill of powdered tissue to a sample tube with a) 180ul lysis buffer T, 20ul proteinase K - GenElute b) 198ul DNA extraction buffer, 2ul proteinase K - ZebraFish § So the final concentration of proteinase K were a) 2mg/ml and b) 200ug/ml § Samples were kept in a shaking water bath at 50-55 degrees • 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. • We measured the DNA content of supernatant from the Kit extraction with nanodrop spectrophotometer - sample from GenElute taken: ○ 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) • Template used in all: L.opalsecns genomic DNA A1 tube extracted with DNA Kit from Sigma and different sets of primers: ○ A1 ○ A2 ○ B1 ○ 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

LCO1490: 5'-ggtcaacaaatcataaagatattgg-3' HC02198: 5'-taaacttcagggtgaccaaaaaatca-3

• Picture of PCR - again no trace of amplified rflectin - no detectable primers and template DNA ○ Order of samples 1:A1, 2:A2, 3:B1, 4:+, 5:Ladder IV ○ No visible bands in the gel (apart from the Ladder IV) ○ Positive control does not work - although in the cited paper, successful use in Loligo pealei is shown ○ Suggests that the prepared DNA was not sufficiently clean • As we read in some papers (reference), difficult to work with genomic DNA from squids and other cephalopods.