| Cloning MultiPlex PCR-based Products for DNA Sequencing in pUC-based Vectors | | 点击： 作者：51protocol收集 来源： 时间： 2007-03-25 本站论坛 |
|  | Cloning Multiplex-PCR Products into pUC Vectors for DNA Sequencing
Latest update 2-21-00
- PCR Product Clean-up:
- End-repair and phosphorylation ( directly from Roe protocol book)
- DNA ligation (from Roe Lab protocol book)
- Transformation (from Roe Lab protocol book)
- Subclone isolation (from Roe Lab protocol book)
- Add the following to each reaction:
- 10 units of USB shrimp alkaline phosphatase (SAP)
- 100 units of USB exonuclease I
- Incubate for 30 minutes at 37 degrees C, 10 minutes at 80 degrees C
- Store at 4 degrees C until further analysis
- Check PCR products on 1% agarose gel containing ethidium bromide (reference Roe Lab protocol book) loading 5-10 ml of the reaction products
- Extract the remaining sample once with an equal volume of phenol/chloroform (1:1)
- Precipitated with 2.5 volumes of ethanol/acetate at -20 degrees C overnight
- Wash with 70% ethanol
- Dry and resuspend in 30 ml of sterile distilled deionized water.
- Add the following to each reaction:
* 10X Kinase buffer 5 ml
* 10 mM rATP 5 ml
* 0.25 mM dNTPs 7 ml
* T4 polynucleotide kinase 1 ml (3 U/ml)
* Klenow DNA polymerase 2 ml (5 U/ml)
- Incubate at 37 degrees C for 30 minutes.
- Phenol/chloroform extract as above.
- Precipitate with ethanol-acetate as above
- Wash with 70% ethanol.
- Resuspend in ~15 ml sterile distilled deionized water.
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