T载体自制法！
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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 it's 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 won't damage the DNA, some even add Guanine to 10 mM to protect the DNA from the damage effect of UV light), retract the band. Resuspend in 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).
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 it's 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 won't damage the DNA, some even add Guanine to 10 mM to protect the DNA from the damage effect of UV light), retract the band. Resuspend in 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).
