Great ligation problem - Double digested DNA insertion problem (Nov/20/2007 )

Dear all
I am trying to insert 1.2kb DNA fragment into a vector whose size is closed to 4kb. I used XhoI and SacII for the insert and BamHI and SacII for the vector. I make dephosphorylation of both ends of the insert. And BamHI end of the vector also dephosphorylated by SAP. (During insert dephosphorylation I did not use SAP buffer.) Now I want to do ligation between the insert and vector through SACII region. I tried for 6 months but everytime I have problems to get nice DNA band near the target size. But recently I got this website address. I hope it will be very helpful. After ligation with T4 DNA ligase I am always getting a faint band near 5 kb but this amount is too low that I cant use this for the growth in competence cell. Yesterday I got dimer and trimer of the insert during ligation. But the target band is still very faint.

I am confused why I am not able to get nice band of the target size. Please reply and help me.

To replicate in a cell, the DNA must be recircularized. If you are dephosphorylating both the insert and vector, the ligation can never happen. One end or the other must have a 5' phosphate to ligate.

Hi,

as far as i knw u need to dephos. either the vector or the insert not both. and mostly its the vector which is dephosp so u could use the same for any other insert other than the one in current use

Why are you wanting a linear DNA? Is that standard for yeast transformation?

As phage434 mentioned, dephosphorylating both your insert and vector is a bad thing. For ligation to occur one molecule has to carry the 5' phosphate. If neither molecule does, then ligation can not occur. This is probably why you don't see much on your gel, only the few escapees from the dephosphorylation reaction can ligate.

Also ....umm... you also realise that BamHI and XhoI do not naturally form compatible ends. Only partial filled in BamHI and Xho (2bp partial fill) will actually ligate. Which means your vector-insert complex can not recircularise and thus propagate in bacteria for amplication.


What you want to do is to
1- cut your vector with BamHI
2- then partial fill in the BamHI site with 2bp using Klenow and the appropriate dNTP
3- clean up using a column or via phenol/chlorofrom
4- digest with SacII
5-gel purify the vector

6- cut the insert with XhoI
7- then partial fill in the XhoI site with 2bp using Klenow and the appropriate dNTP
8- clean up using a column or via phenol/chlorofrom
9- digest with SacII
10- gel purify the insert

Put vector and insert together and ligate (dephosphorylation of the vector is not required)
Transform your DNA into cells, select the correct colony either by colony PCR or by minipreping colonies and testing by restriction digest.

Get the colony that you want and midiprep it. Making lots of DNA.