Volume 30 Issue 5
Sep.  2013
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ZHOU Shengcai, HUANG Huahong, TONG Zaikang, WU Xiaolin. Optimization of an AFLP protocol with four Phoebe species[J]. Journal of Zhejiang A&F University, 2013, 30(5): 789-796. doi: 10.11833/j.issn.2095-0756.2013.05.024
Citation: ZHOU Shengcai, HUANG Huahong, TONG Zaikang, WU Xiaolin. Optimization of an AFLP protocol with four Phoebe species[J]. Journal of Zhejiang A&F University, 2013, 30(5): 789-796. doi: 10.11833/j.issn.2095-0756.2013.05.024

Optimization of an AFLP protocol with four Phoebe species

doi: 10.11833/j.issn.2095-0756.2013.05.024
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  • Corresponding author: TONG Zaikang
  • Received Date: 2012-10-08
  • Rev Recd Date: 2013-03-12
  • Publish Date: 2013-10-20
通讯作者: 陈斌, bchen63@163.com
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    沈阳化工大学材料科学与工程学院 沈阳 110142

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Optimization of an AFLP protocol with four Phoebe species

doi: 10.11833/j.issn.2095-0756.2013.05.024
    Corresponding author: TONG Zaikang

Abstract: To meet the needs of an amplified-fragment-length-polymorphism(AFLP) protocol,high quality genome DNA was extracted from Phoebe chekiangensis,Phoebe sheareri,Phoebe zhennan,and Phoebe bournei seedlings with improved cetyltrimethylammonium bromide(CTAB)methods. For optimization of restriction system(30.0 L),the genome DNA,300 ng;was buffered with 0.3 L;bovine serum albumin (BSA),0.3 L;EcoR,50 nkat;and Mse,25 nkat;and incubated at 37 C for 4 h;then the restriction enzyme was killed with 65 C for 15 min. The best ligation system(25.0 L) was with 20.0 L of the digestion products;T4 buffer,2.5 L;T4 ligase,666.8 nkat;EcoR adapter,5 pmol;and Mse adapter, 10 pmol;and left at 16 C overnight (10-14 h). The optimal preamplification system (20.0 L) included 2.0 L ligation products;10 buffer,2.0 L;Mg2+,31.25 nmol;Taq DNA polymerase;16.67 nkat;dNTP,4 pmol; pre-amplification primers(E+A),6 pmol;and pre-amplification primers(M+C),6 pmol. Also,the best selective amplification system (20.0 L) was 2.0 L,1/40 pre-amplification products;10 buffer,2.0 L; Mg2+,31.25 nmol;dNTP,4 pmol;Taq DNA polymerase,16.67 nkat;amplification primers(M+CNN),15 pmol;and amplification primers(E+ANN),12 pmol. Moreover,screening with the optimized AFLP reaction system resulted in 13 pairs of suitable amplification primers for AFLP analysis of Phoebe chekiangensis. [Ch,5 fig. 3 tab. 24 ref.]

ZHOU Shengcai, HUANG Huahong, TONG Zaikang, WU Xiaolin. Optimization of an AFLP protocol with four Phoebe species[J]. Journal of Zhejiang A&F University, 2013, 30(5): 789-796. doi: 10.11833/j.issn.2095-0756.2013.05.024
Citation: ZHOU Shengcai, HUANG Huahong, TONG Zaikang, WU Xiaolin. Optimization of an AFLP protocol with four Phoebe species[J]. Journal of Zhejiang A&F University, 2013, 30(5): 789-796. doi: 10.11833/j.issn.2095-0756.2013.05.024

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