|Table of Contents|

Establishment and Optimization of PCR-SSCP Amplification Reaction System in Processing Tomato(PDF)

《北方园艺》[ISSN:1001-0009/CN:23-1247/S]

Issue:
2015年15
Page:
88-91
Research Field:
Publishing date:

Info

Title:
Establishment and Optimization of PCR-SSCP Amplification Reaction System in Processing Tomato
Author(s):
MA Haixin; PANG Shengqun; YAN Lijuan; WANG Bin; WEI Haibin; CHENG Linlin
(College of Agriculture，Shihezi Uniiversity，Shihezi，Xinjiang 832003)
Keywords:
processing tomato; PCR-SSCP; polyacrylamide gel electrophoresis; silver-stained
PACS:
-
DOI:
10.11937/bfyy.201515024
Abstract:
Taking processing tomato as material，single strand conformation ploymorphism (SSCP)technique was used,the effect of many factors such as extraction method of DNA,reaction process of PCR,electrophoretic factors(denaturalization temperature and time,electrophoresis temperature,electrophoresis time,electrophoresis buffer and electrophoresis power),denaturant on SSCP technique was studied.The PCR-SSCP reaction system and reaction process which were high polymorphism detection rate,good repeatability,clear stripe were screened and established.The results showed that adding 5 mol/L NaCl and the first centrifugation speed and centrifugal extraction time was 8 000 r/min,15 minutes could obtain high quality DNA in CTAB buffer when extracting tomato leaves’ DNA using the CTAB method,the amplification program was 94℃ predenature 5 minutes,94℃ denature 1 minutes,52℃ annealing 30 s,72℃ extension of 1 minutes,a total of 29 cycles，final extension at 72℃ 10 minutes.Primer size was 100-300 bp.Denaturalization temperature was 98℃,denaturalization time was 10 minutes,non-denaturing polyacrylamide gel concentration of 8%.Electrophoresis time was 1.5-2.5 hours.No added glycerin,the bands of the single strand DNA were clear and easy to read in processing tomato.

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Last Update: 2015-08-18