涂7体外转录翻译.pptVIP

In vitro transcription and In vitro translation  In vitro transcription  In vitro Transcription 体外得到单一,大量的mRNA 意义 探针--原位杂交 SiRNA RNAI Transcrip十 体外翻译,蛋白表达  In vitro Transcription Antisense ECoRI Cut with ecori Use T-3 polymerase Cut with bamhI Use T-7 polymerase  T75 or T3 5 Element for inl vitro transcription of your gene of interest in the sense orientation using the T7 or T3 RNA polymerase T75Element Gene of interest OR T3 5Element -gene of interest sense mRNA T73 or T3 3 Element for in vitro transcription of your gene of interest in the anti-sense orientation using the T7 or T3 RNA polymerase w0[T73Bme0R回dmeH3Bm anti-sense mRNA  T75′ Element T7 Promoter Primer #z T7 promoter sequence binding site\ 5 PO4 GACTCGTAATACGACTCACTATAGGGCCCTT 3 CTGAGCATTATGCTGAGTGATATCCCGGGAAGCCTTG 5 T73′ Element 5 GACTCAAAGGGCCCTATAGTGAGTCGTATTACGAGTC 3 TCCCGGGATATCACTCAGCATAATGCTCAG PO4 5 T7 Promoter Primer #2 T7 promoter sequence  Vector Maps acZ pBluescript ll SK (+)CS Bluescript II SK (+)Multiple Cloning Site Region (sequence shown 59 TTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCC CCCTCGAGGTCGAC ATAAGCTTGATATCGAATTCCTGCAGCC GATCCAC TAGTTCTAGAGCGGCCGC CACC GCGGTGGAGCTC CAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGT AATCATGGTCATAGCTGTTTCC  In vitro transcription Plasmid with T3, T7 or SP6 promoters Linearization of plasmid dna by restriction enzyme In vitro transcription: Plasmid buffer NTP labeled UTP RNa polymerase DNASe digestion, Phenol/ chloroform extraction and RNA precipitation  In vitro transcription reaction Gloves should be worn at all times during preparation of in vitro RNA. All solutions should be rNase-free i.e. made with depc water if home-made or bought specifically to use for rNa work  In vitro transcription reaction You will need Ribonuclease inhibitor(RNasin) T7 or T3 RNA Poly merase 2.5mMrNTPs x T7/T3 RNA Polymerase Buffer(200mM Tris Cl pH8.0 125mM NaCl, 40mM MgCl2, 10mM spermid