IDENTIFICATION OF COPPER-INDUCIBLE GENES IN Pistia stratiotes

More documents

Recommendations

Info

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 v
3.16.3.4 Reverse transcription of mRNA 54 3.16.4 Amplification of 5’ cDNA end 54 4.0 RESULT 57 4.1 Determination of copper absorption by Pistia stratiotes 57 4.2 Identification of copper-inducible gene fragments in 59 Pistia stratiotes 4.2.1 Isolation of total cellular RNA from Pistia stratiotes 59 4.2.2 Amplification of copper-inducible candidate gene 61 fragments 4.2.3 Cloning of copper-inducible candidate gene 63 fragments 4.2.4 Sequence analysis of copper-inducible candidate 65 genes 4.3 mRNA expression study of copper-inducible candidate 65 gene fragments 4.3.1 Determination of the specificity of primers and 72 conditions for amplification 4.3.2 Dose response analysis on the mRNA expression 74 levels of copper-inducible candidate genes in copper- treated Pistia stratiotes 4.4 Amplification of the 5’ end of the full length candidate gene 76 fragment 1 mRNA from Pistia stratiotes 4.4.1 RACE-PCR 76 4.4.2 Cloning and sequencing of the RACE-PCR product 79 vi
Page 1 and 2: IDENTIFICATION OF COPPER-INDUCIBLE
Page 3 and 4: ACKNOWLEDGEMENT First of all, I wou
Page 5: 2.8 The use of living organism to m
Page 9 and 10: LIST OF FIGURES 3.1 Vector used in
Page 11 and 12: LIST OF TABLES x Pages 2.1 The role
Page 13 and 14: LIST OF PLATES 2.1a Side view of Pi
Page 15 and 16: HNO 3 Nitric acid IPTG Isopropyl-β
Page 17 and 18: IDENTIFICATION OF COPPER-INDUCIBLE
Page 19 and 20: 1.0 Introduction For many years, th
Page 21 and 22: ioindicator characteristic after be
Page 23 and 24: and may identify potential diagnost
Page 25 and 26: Plate 2.1a. Side view of Pistia str
Page 27 and 28: 2.3 Effects of heavy metals on the
Page 29 and 30: decreasing red cell survival in car
Page 31 and 32: and efficient functioning of the bo
Page 33 and 34: The suggested safe level of copper
Page 35 and 36: uild-up of toxic concentrations at
Page 37 and 38: 2.7 Toxicity Test Toxicity test is
Page 39 and 40: 2.8.1 Bioindicator Adam (1990) defi
Page 41 and 42: This technique makes use of biologi

IDENTIFICATION OF COPPER-INDUCIBLE GENES IN Pistia stratiotes

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?