T-载体的制备方法.docVIP

?1　用平末端限制性内切酶切载体，再用一步PCR法在平末端加T；2　用带T的粘性末端限制性内切酶切载体，然后用末端加尾法（除dNTP外）产生T末端；3　合成可用限制性内切酶产生T末端的核苷酸短链，插入载体中用限制性内切酶酶切即可。　 ? Making the T-vector:1. Digest 5 ug of vector DNA ( pBluescript ＆ pUC18) with a restriction enzyme that generates a unique blunt end site, for example EcoRV or Sma I ( we prefere EcorV for its stable activity at 37 C) for 2 hours at 37 C.2. Run the digest on a 1% low-melting-point agarose (use TAE as buffer, because the boric acid in TBE may inhibit the ligation step). Excise the vector DNA under UV ( use long wave UV source, so you wont damage the DNA, some even add Guanine to 10 mM to protect the DNA from the damage effect of UV light), retract the band. Resuspendin a volume of 20 ul of water in a 0.5 ml eppendorf tube.3. Add 5 ul of 10X PCR buffer, 1 ul of 100 mM dTTP, 24 ul of distilled water and 0.4 ul of Taq DNA polymerase. Overlay with 40 ul of mineral oil. Incubate at 72 C for 2 hours.4. Purify the T-vector by phenol/chloroform extraction and ethanol precipitation. Resuspend the prepared T-vector in 100 ul of water or TE, giving a concentration of 50 ng/ul.(You better check the concentration yourself using any method you are confortable with). ? Making the T-vector（another）: T-vectors facilitate the cloning of DNA fragments that have been generated by the Polymerase Chain Reaction (PCR). Taq Polymerase adds a single deoxyadenosine (A) nucleotide to the 3 ends of the PCR products. This activity is not template dependent. The T-vector generated in this protocol contains a single deoxythymidine (T) residue at the 3 ends. This T residue on the vector will hybridize to the single A overhang on the PCR product and increase the efficiency of ligation. Procedure:1. Digest 1 μg of plasmid (such as pBlueScript SK-) with a blunt-end restriction enzyme??????? ?(such as EcoR V) as follows:? 1 μg of DNA in 40 μl of ddH2O? 5 μl of 10X EcoRV Restriction Digest Buffer? 0.5 μl of EcoRV Restriction Enzyme? Adjust the final volume to 50μl with ddH2O?