|
Tuesday, November 18, 2003
Description Quick Change Mutagenesis Procedure 1. Set up two separate primer extension reactions (one for each top and bottom primer) containing: 5 microliters 10X Pfu Buffer (supplied with enzyme) 1 microliter 10 micromolar primer (0.13 microgram 45-mer) 0.1 0.2 microgram plasmid template 1 microliter 10 mM dNTP mix H2O to a final volume of 50 microliters Add 1 microliter Pfu turbo polymerase (Stratagene) Incubate: 1. 94 deg, 30 sec 2. 95 deg, 30 sec 3. 55 deg, 1 min 4. 68 deg, 2 min/kb up to 10 KB plasmid Repeat steps 2-4 for a total of 4 cycles Hold at 4 deg. Combine 25 microliters from each extension reaction above. Add 1 microliter Pfu polymerase. Incubate as above, except repeat a total of 18 cycles. Remove 25 microliters of the reaction. Add 10 units of DpnI enzyme. Mix well and incubate at 37 deg for at least one hour. Transform 1 microliter of the reaction to electrocompetent cells. Plate 100 and 20 microliters of cells on separate plates. Expect ~100 colonies on the 20 microliter cell plate. Recipes Supplies Tips (责任编辑：泉水) |
Quick Change Mutagenesis
时间:2005-07-18 00:00来源:Scienceboard.net 作者:admin
顶一下
(0)
0%
踩一下
(0)
0%
------分隔线----------------------------
- 发表评论
-
- 最新评论 进入详细评论页>>
