|
PCR Protocol 10X PCR Buffer 1.0 MgCl2 (2.0mM) 0.8µl dNTPS mix* 0.8µl Primer-F (µg/µl) 0.1µl Primer-R (µg/µl) 0.1µl Taq (5U) 0.1µl 32P dCTP (10µci/µl) 0.1µl ddH2O 4.5µl DMSO (100%) 0.5µl SUBTOTAL 8.0µl Template DNA (50-100ng) 2.0µl Note: Synthesize primers 21-30 nt in length for products of 200-350 bp. Use standard PCR conditions, however the Tm has to be calculated from the specific primer pair. *dNTPS mix (final concentration): dATP 2.5mM; dTTP 2.5mM; dGTP 2.5mM; dCTP 1.25mM SSCP Gels Prepare 0.5x MDE gel as follows: MDE gel 16.0ml ddH2O 44.2ml 10X TBE 3.84ml 10% APS 256µl TEMED 25.6µl Pour sequencing gel format with appropriate sharkstooth comb. Gel will polymerize in about 1 hour. Loading Buffer 95% formamide 10mM NaOH 0.025% Bromophenol Blue 0.025% Xylene Cyanol Run gel in 0.6X TEB buffer. Heat denature samples at 94°C for 5 minutes and place them on ice for 3-5 minutes. Load 2.0-4.0µl per sample. Include non-denatured controls. Electrophoresis conditions Fragment Size: 150-200 bp 6 Watts 10-12 hours room temperature Fragment Size: > 200 bp 8 Watts 10-12 hours room temperature Exposure Dry gel and expose either at -80°C for 2 hours or at room temperature for 16-18 hours |
→如果您认为本词条还有待完善，请 编辑词条
上一篇ES injection pipette manufacture（图） 下一篇Collection of Morulae and Earlier
词条内容仅供参考，如果您需要解决具体问题
（尤其在法律、医学等领域），建议您咨询相关领域专业人士。
0
收藏到: 
