40bp寡核苷酸连接如何连接到载体上？...

自己顶起来，问题还没有解决，有经验的哥哥姐姐们，求分享！

Try to run native PAGE to purify the two 40 mer oligomers. Have the two purified 40 mer oligomers (2 um each) reannealed. DO NOT cut with enzyme, and ligate with vector which has been cut with EcoRV, or Sma I, or Stu I, or cut with any unique enzyme and been blunt ended. Set ligation at room temperature and incubate the ligation overnight. Make 1:3 dilution of the ligation with sterilized ddH2O, and use 3-5 ul of the diluted ligation for transformation. Make 12-15 miniprepes and run DNA sequence to identify the positive clone.

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mpark

Try to run native PAGE to purify the two 40 mer oligomers. Have the two purified 40 mer oligomers (2 um each) reannealed. DO NOT cut with enzyme, and ligate with vector which has been cut with EcoRV, or Sma I, or Stu I, or cut with any unique enzyme and been blunt ended. Set ligation at room temperature and incubate the ligation overnight. Make 1:3 dilution of the ligation with sterilized ddH2O, and use 3-5 ul of the diluted ligation for transformation. Make 12-15 miniprepes and run DNA sequence to identify the positive clone.

谢谢您的建议，我想问一下您几个问题。

1. 在将40 mer的寡核苷酸退火时，需不需要磷酸化？退火缓冲液直接用水可以吗？需不需要加Nacl， EDTA, Tris-Cl等？

2· 您说的连接反应是在室温连接过夜吗？25℃条件下过夜会不会有影响，我之前查了相关资料，有老师说室温左右连接最好不要超过3个小时，不然寡核苷酸末端会丢失影响连接效果，会不会存在这样的问题？

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Do not have the oligomers phosphorylated. Add 5 M naCl in reannealing solution to a final of concentration 150-200 mM.

25 C ligatino overnight is perfect. If the oligomers are not purified, it could happen for poor ligation, but not due to nucleotides broken away, instead due to non-blunt ended double-stranded oligomers.

.

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mpark

Do not have the oligomers phosphorylated. Add 5 M naCl in reannealing solution to a final of concentration 150-200 mM.

25 C ligatino overnight is perfect. If the oligomers are not purified, it could happen for poor ligation, but not due to nucleotides broken away, instead due to non-blunt ended double-stranded oligomers.

.

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谢谢mpark的建议。

我连过一个40bp的片段，不过，用的是无缝克隆的方法。阳性率高，推荐你用，至于试剂盒，我就不打广告了，一搜一大片。别买进口的就行。太贵

个人认为寡核酸片段太稀了吧，我们做接头时弄的浓度是50pmol/ul,稀释1pmo左右连，搞不清你为啥要酶切，设计时不能弄成突出末端吗

lqz816

个人认为寡核酸片段太稀了吧，我们做接头时弄的浓度是50pmol/ul,稀释1pmo左右连，搞不清你为啥要酶切，设计时不能弄成突出末端吗

您好，谢谢您的答复，按照你说的最终连接浓度时1 pmol/ul 即1 umol/L的浓度。而我之前的操作到连接反应时的你那浓度时0.5 pmol/ul。所以应该跟您说的浓度差不多，但是的确有点稀。

我做酶切是因为我在设计oligo 时在末尾加了酶切位点跟保护碱基，我怕不酶切会影响它与酶切后线性载体的连接，就先进行了酶切，在通过高温孵育的方式将酶灭火。

future007

我连过一个40bp的片段，不过，用的是无缝克隆的方法。阳性率高，推荐你用，至于试剂盒，我就不打广告了，一搜一大片。别买进口的就行。太贵

哦哦，没听说可以用试剂盒，可以私信一下您用的是哪个公司的试剂盒吗？

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细胞君爆发中

lqz816

个人认为寡核酸片段太稀了吧，我们做接头时弄的浓度是50pmol/ul,稀释1pmo左右连，搞不清你为啥要酶切，设计时不能弄成突出末端吗

您好，谢谢您的答复，按照你说的最终连接浓度时1 pmol/ul 即1 umol/L的浓度。而我之前的操作到连接反应时的你那浓度时0.5 pmol/ul。所以应该跟您说的浓度差不多，但是的确有点稀。

我做酶切是因为我在设计oligo 时在末尾加了酶切位点跟保护碱基，我怕不酶切会影响它与酶切后线性载体的连接，就先进行了酶切，在通过高温孵育的方式将酶灭火。

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可以在设计接头时把酶切位点互补序列设计成悬挂/突出末端，这样可免片段酶切步骤，直接退火后连到酶切好的载体上，有时加保护碱基酶切效率不见得就高