网站地图	本站论坛
高级搜索	收藏本站

热门关键字：　细胞培养　 pcr 　琼脂糖凝胶电泳　质粒DNA的提取　凝胶加样缓冲液　 PCR inventor

	当前位置：试验方案>核酸试验>PCR>tail-pcr> 正文

TAIL-PCR to clone flanking sequence from Feldmann

点击： 作者：51protocol收集 来源： 时间： 2007-03-29 　本站论坛

Thermo-cycling: #10 to #18, and then #19, #20, #21, #53
查看大图

Cycling #10: 92^oC; 5 min;[Add 0.2 ul Taq polymerase (Perkins-Elmer's or Stratagene's) after 4 min denaturation, link to clcling #18
查看大图

Cycling #18 (5 cycles of the following):

查看大图

94^oC, 1'

62^oC, 1'

72^oC2.5'

Cycling #19 (1 cycle of the following):

查看大图

94^oC, 1'

25^oC, 3'

Ramp to 72^oC in 3'
查看大图

72^oC2.5'

Cycling #20 (15 cycles of the following):

查看大图

94^oC, 30''
查看大图

68^oC, 1'

72^oC, 2.5'
查看大图

94^oC, 30''
查看大图

68^oC, 1'

72^oC; 2.5'
查看大图

94^oC, 30''
查看大图

44^oC; 1'

72^oC; 2.5'

查看大图

cycling #21:

查看大图

72^oC, 5'

Cycling #53

查看大图

4^oC. soak

Dilute the primary reaction product with water to 1/50, then set up the 2nd reaction

查看大图

44ul PCR stock
查看大图

2.5ul TL-2 (50uM)
查看大图

2.5ul AD-2 or OPA-2 (50uM)
查看大图

1ul 1/50 diluted 1st reaction

Thermo-cyclings: #10 (0.2u. Taq polymerase was added after 4 min denaturation) to #22, and then #21, #53.
Cycling #22 for 12 cycles:

查看大图

94^oC, 30''
查看大图

64^oC, 1'

72^oC, 2.5'
查看大图

94^oC,30''
查看大图

64^oC, 1'

72^oC, 2.5'
查看大图

94^oC, 30''
查看大图

44^oC, 1'

72^oC, 2.5'

Dilute the 2nd PCR product to 1/50 with water, and carry out the tertiary reaction

查看大图

44ul PCR stock
查看大图

2.5ul TL-1
查看大图

2.5ul AD-2 or OPA-2 (50uM)
查看大图

1ul 1/50 diluted 2nd reaction product

Thermo-cycling: #10 (0.2ul Taq polymerase was added after 4' denaturation in #10), and then #12, #52 .
Thermo-cycling #12 for 25 cycles:

查看大图

94^oC,1'

37^oC, 1

72^oC, 1'

1.5% agarose electrophoresis of all three PCR reaction products.

Purify the PCR products using Pharmacia's Spin-Column S-300.

Cloning PCR product by using Stratagene's PCR polishing kit and pCRScript Amp (SK ) cloning kit or my favorite one-tube cloning method .

上一篇：A Problem of TAIL PCR 下一篇：Real-time PCR 专题