View stunning SlideShares in full-screen with the new iOS app!Introducing SlideShare for AndroidExplore all your favorite topics in the SlideShare appGet the SlideShare app to Save for Later — even offline
View stunning SlideShares in full-screen with the new Android app!View stunning SlideShares in full-screen with the new iOS app!
Homogenization and Lysis [ 1.4 M sodium chloride, 1% pvp, 50.0 mM EDTA, 2% CTAB, 2% β - mercaptoethanol] .
Protein and RNA digestion
Peptide precipitation( 5 M potassium acetate)
Chloroform extraction :- Samples were extracted with equal volume of phenol : CHCl 3 : isoamyl alcohol (25:24:1)
NaCl containing PEG was added to increase the DNA yield. Precipitation of DNA and washing
Obtained pellet was dissolved in 60.0 µl of TE buffer [10 mM Tris (pH 8.0),
0.1 mM EDTA] and stored at 20º C until use.
It was not possible to extract DNA from processed tea leaf samples even by using modified CTAB method. Another extraction method described by Hupfer et al ., (1998); Hotzel et al ., (1999); Meyer et al ., (1997); Poms et al ., (2001) was used to extract DNA from processed tea leaves. Processed tea leaves and tea dust were used to extract DNA .
Removal of Contaminants CTAB precipitation solution (5 g/L CTAB, 0.04 M NaCl)
DNA Precipitation and washing
Pellet was dried and re-dissolve in 100.0 µl of sterile deionised water.
Selection of a Barcoding region The trnH – PsbA spacer (~450 bp) of the plastid was selected as the barcoding region. This region is one of the most variable and easily amplified across a broad range of land plants.
W for A or T Y for C or T primer Sequence(5’-3’) Annealing temperature (ºC) Expected Product Size (bp) Forward primer Trn – H CGCGCATGGTGGATTCACAATCC 50 450 Reverse Primer Psb – A GTWATGCAYGAACGTAATGCC
PCR Agarose gel electrophoresis and gel purification Sequencing and editing
2% agarose gel stained with ethidium bromide showing PCR amplified products of DNA extracted from fresh leaf sample using modified CTAB method. 450 bp marker
Lanes 1 – 16. DNA extracted from samples TRI 3063, TRI 3022, TRI 4006, TRI DN, TRI 4014, TRI 4021, TRI 3015, TRI 3018, TRI 4042, TRI 4052, TRI 2025, TRI 4028, TRI 2043, TRI DG7, TRI 3025, TRI 4046, respectively. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16