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3.5 Construction and screen of subtractive library
To set up the ligation reaction of PCR products and
vector, a reaction was done with 3 µL recovered PCR
products and 1 µL pGEM-T easy following instruction
of the pGEM-T easy kit. 2 µL ligation reaction products
were transformed into
E. coli
super competent cells by
heat shock, transformed cells were plated on a LB
Patri dish. Cells grew overnight. Once white-blue clone
visible, clones were screened by strain PCR with TSH
primers. Positive clones were selected by gel electrop-
horesis and confirmed by sequencing (ABI 3730XL
DNA Sequencer at the Shenggong Gene Center,
Shanghai, China).
3.6 List of abbreviations
PCR: polymerase chain reaction; RT: reverse transcripttion;
TSH: transcriptome suppression subtractive hybridization;
SSH: suppression subtractive hybridization; pC-SG: a
recombinant plasmid contained 6 genes as shown in
the table 1; JM109: an
E. coli
strain; JM109
-
: JM109
without any plasmid; JM109
+
:JM109 contained plasmid
of pC-SG: Other abbreviations as shown in the table 1
and table 2.
Authors' contributions
XLY designed this experiment, primers and adaptor, and wrote
and revised the manuscript; YJM performed subtractive
hybridization, amplification of tester specific cDNA and
establishment of tester specific cDNA library; YJM, ZPH and
QW contributed to literature retrieve, BLAST search and ESTs
sequence analysis. All authors read and approved the final
manuscript.
Acknowledgements
This work was jointly supported by National Sci-Tech Support
Program in China (2009BADC5B01, to XY) and National High
Tech-Dev Program (863) in China (2006AA100107, to XY).
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