IDENTIFICATION OF COPPER-INDUCIBLE GENES IN Pistia stratiotes

3.10.2 Amplification of the first cDNA strand 39
3.11 Cloning of PCR products 42
3.11.1 Extraction of DNA from agarose 42
3.11.2 Assessment of the concentration of the purified 43
PCR fragments
3.11.3 Optimizing insert:vector molar ratio 43
3.11.4 Ligation of PCR fragments to pGEM ® -T Vector 43
3.11.5 Preparation of competent cells 44
3.11.6 Transformation of competent cells 44
3.11.7 PCR-screening of recombinant colonies 45
(colony PCR)
3.12 Isolation of recombinant plasmid 45
3.12.1 Wizard ® Plus SV Minipreps DNA Purification System 45
3.13 Restriction endonuclease digestion of DNA 47
3.14 Sequencing of the PCR products 47
3.14.1 Sequence analysis 47
3.15 Gene expression study 47
3.16 RNA ligase-mediated and oligo-capping rapid amplification 48
of cDNA ends (RLM-RACE)
3.16.1 Isolation of total cellular RNA 48
3.16.2 DNase treatment of RNA 48
3.16.3 Synthesis of full-length of 5’ cDNA ends 51
3.16.3.1 Dephosphorylation of RNA 51
3.16.3.2 Removal of mRNA cap structure 51
3.16.3.3 Ligation of GeneRacer TM RNA Oligo 52
to decapped mRNA
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