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troubles to make a 3 ways DNA ligation

troubles to make a 3 ways DNA ligation - Molecular Cloning Forum

troubles to make a 3 ways DNA ligation - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 09-14-2010, 11:59 PM
Pipette Filler
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Default troubles to make a 3 ways DNA ligation



I have troubles to make a 3 ways DNA ligation

My setup is

1] Vector pEt 15b digested with Nde1/Xho1 and dephosphorylated and cleaned up with qiagen kit
2] Insert 1, 210 bp from pcr product digested with Nde1/Bamh1 and cleaned up with qiagen kit
3] Insert 2, 210 bp from pcr product digested with BglII/Xho1 and cleaned up with qiagen kit too
I have tried a molar ratio vector/insert 1/3 without success
Have you some advices for me
Thanks
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