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TitleSTANDARDIZATION OF PLANT TISSUE CULTURE AND TRANSFORMATION PROTOCOLS FOR
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Table.3.4: Solutions used in alkali lysis method of plasmid DNA isolation

Chemical Components Final Conc.

Solution I (pH 8.0) Glucose 50 mM

Tris (pH 8.0) 25 mM

EDTA (pH 8.0) 10 mM

Solution II SDS 1.0 %

NaOH 0.2 N

Solution III Ammonium acetate 7.5 M

TE Buffer Tris (PH 8.0) 1.0 M

EDTA (pH 8.0) 0.5 M



3.14. PCR analysis

The initial screening for the presence of transgene in regenerated plants was done

using PCR technique as per the methods described by Jain et al., (1999).Plant DNA

isolated from leaf tissue was used as template to obtain amplification product of mVIP

and mcryIAc genes for screening of putative transgenic plants. For integration of trans

gene, following gene specific primer pairs were used.

mcryIAc primer

Forward-5‟ATG GAT AAC CCA AAC ATT AAC-3‟

Reverse-5‟GTA CTC AGC CTC AAG AGT GGC-3‟

mVIP primer

Forward-5‟GTT GAC CAC TAG AGC TTT GC-3‟

Reverse-5‟CTT AAT AGA GAC ATC GTA G-3‟

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