菌落pcr资料（Colony PCR data）.docVIP

菌落pcr资料（Colony PCR data） Colony PCR (Colony, PCR) method Colony PCR (Colony, PCR) does not need to extract genomic DNA, without enzyme cutting identification, but directly uses the DNA exposed after bacterial pyrolysis as template for PCR amplification, with less time and effort. Universal primers are recommended for use on vectors. This method is usually used for recombinant screening or DNA sequencing analysis. The final size of the PCR product is the size of the insertion fragment between the vectors and the universal primers. Concrete method: Preparation of mixtures of 1 and PCR Taq buffer (10 x) 20 UL DNTP (2.5, mM) 5 UL Primer Forward (50 mM) 10 UL Primer Reverse (50 mM) 10 UL DdH2O 147, UL Taq (2U/ul) 8 UL Total 200, UL Mix the solutions together. 10 UL each tube is packed in a 200 UL PCR tube. Transformants on the board into 2 randomly selected, under normal temperature, with a toothpick or gun head of a cell were sterilized, click on LB agar plate, make a copy; and then stained with a toothpick or gun head on the cell corresponding with PCR mixture of PCR tube washing number (good mark, pipe if the flat point is No. 1, No. 1 is also marked on the tube, so as to select to clone after expanding to the culture, tightly tube). 3. The PCR mixture mixed with bacteria was placed in a PCR instrument and amplified according to conventional conditions. Did the electrophoresis test get the target fragment?. If yes, positive clones. 4. The plates inoculated with colonies were incubated in 37 incubator for the night, and the colonies were amplified. The positive clones were selected for screening or culturing the next day. Step 2 can also not point board, direct inoculation of shake, and if the morning to do PCR, then in the afternoon can be improved grain, get recombinant vector. Note: the design of primers is critical. In general, if directed cloning, the use of universal primers on the carrier can; such as pET series can be used T7 universal primers. If it is a non d