RT-PCR Analysis Solutions 10X RT Buffer 10X PCR Buffer 100 mM Tris pH 9.0 500 mM KCl 1% Triton X-100 25 mM MgCl 2 use at a concentration of 1.5 mM Lysis Solution 4M GuSCN 250 g guanidine thiocyanate 25mM Na citrate 7.0 17.6 ml 0.75M Na citrate pH 7.0
DNA 测序原理和方法 DNA 序列测定分手工测序和自动测序，手工测序包括 Sanger 双脱氧链终止法和 Maxam-Gilbert 化学降解法。自动化测序实际上已成为当今 DNA 序列分析的主流。美国 PE ABI 公司已生产出 373 型、 377 型、 310 型、 3700 和 3100 型等 DNA 测序仪，其中
1. Run the gel as normal. Often for genomic southerns it is desirable to run long gels (18cm) over 4-6hrs. 2. Photograph the gel with a ruler adjacent to the molecular weight markers as a reference. 3. Alkali transfer buffer = 0.5M NaOH (20g/lt), 1.5
BUFFERS: 1) Prehybridization/hybridization buffer 1% SDS 6X SSC 10 % dextran sulfate 100 mg salmon sperm DNA/ml (added with the radioactive probe) Note: Store hybridization buffer at 4 degrees and warm to 65 degrees prior to use since the dextran su
This protocol works very well with Hybond-XL membrane from GE Healthcare (formerly Amersham). 1) Digest your genomic DNA, using about 10 μg per reaction in 100 μL total digestion volume. Digest overnight,spiking with a bit more enzyme for 2 more h
Solutions Protocol: Digest 5-10 μg genomic DNA overnight with restriction enzyme of choice. Run digested gDNA on 0.8% TAE gel with marker (with no ethidium bromide) Transfer Setup: 1. Remove gel and place into newly made EtBr solution and rock for
Materials: Whatman 3 mm Blotting Paper nitrocellulose (Schleicher Schuell, Amersham) or nylon membrane filter (Amersham). Paper towels (preferably C-fold "cheap-o" variety) Pyrex or Tupperware dish, glass plates and rubber stoppers A weight (lead pi
1. Run gel (0.8 -1.0% agarose is best). For yeast chromosomal Southerns, digest 20 μg DNA. Use 50 ml minigel for most purposes. Photograph gel, but minimize exposure to UV light. 2. Depurination: Place gel in 250 mls 0.25M HCl.(250 mls = 11 mls 5.8
