double-digest cloning problem - cloning (May/25/2006 )
Dear all,
I need some help in cloning.
I'm trying to insert a 3.4kb fragment into my 5.3kb vector. Here is some information of my procedure.
The fragment is from PCR and double-digested by Nde1 and Sal1(NEB), and heat-inactivated at 65C for 20min.
The vector is also cutted by Nde1 and Sal1, heat-inactivated, and then dephosphatased by Antarctic Phosphatase from NEB.
The molar ratio of the ligation is 1:3 (vector: insert). 16C, overnight.
Competent cells are One Shot® OmniMAX™ 2 T1 Phage-Resistant Cells from Invitrogen. I took 5ul ligation reaction for transformation. Finally, I got 10 colonies.
But, the problem is that all of the plasmids from these ten colonies are the same size as the no-insert vector. That means all of them are self-ligated vectors.(ps, I didn't get 0 colonies from no insert ligation.)
I have dephosphatased the vector and it supposes to be no self-ligation, right?
Do you guys know what maybe the problems?
Thanks a lot!
I need some help in cloning.
I'm trying to insert a 3.4kb fragment into my 5.3kb vector. Here is some information of my procedure.
The fragment is from PCR and double-digested by Nde1 and Sal1(NEB), and heat-inactivated at 65C for 20min.
The vector is also cutted by Nde1 and Sal1, heat-inactivated, and then dephosphatased by Antarctic Phosphatase from NEB.
The molar ratio of the ligation is 1:3 (vector: insert). 16C, overnight.
Competent cells are One Shot® OmniMAX™ 2 T1 Phage-Resistant Cells from Invitrogen. I took 5ul ligation reaction for transformation. Finally, I got 10 colonies.
But, the problem is that all of the plasmids from these ten colonies are the same size as the no-insert vector. That means all of them are self-ligated vectors.(ps, I didn't get 0 colonies from no insert ligation.)
I have dephosphatased the vector and it supposes to be no self-ligation, right?
Do you guys know what maybe the problems?
Thanks a lot!
I dont think you need to dephosphorylate while you have two different sites at two ends. It is possible that the 10 colonies came from:
1- Self-ligation between vector and it own sequence when you excised (as you mentioned that after digesting vector you just inactivated by heat)
2- The remaining uncut vectors from your digestion and and the ligation between your insert and vector did not work (this could be due to inhibition or so) - More likely this case
I am having trouble with ligation myself, but my case is that I tried to ligate 2 PCR fragments together and to the vector. It does not work so far! (it seems like, first 2 fragments ligate together to form a single insert, then it ligate to the vector).
Did u purify the cut vector and PCR on gel to purify them. If not, then better do it, as it can help to avoid minor contaminations.
Recently I also had problems with salI digestion. Try digesting with NdeI and then do it with sal1. It worked for me.
What is the total volume of ligation mixture? Bcoz somehow adding 5ul of ligation reaction seems too much.
Good Luck !!!
Hi, dtle, Scolix,
Thank you very much for your suggestions!
To Dtle,
"and the ligation between your insert and vector did not work (this could be due to inhibition or so) - More likely this case"
---I'm a little confused. What kind of inhibition do you mean? the wrong molar ratio? or the insert is too much?
To Scolix,
The total volume of ligation mixture is 20 ul, in which there are 50ng vector and 96ng insert.
Then how many ligation reaction should I add to competent cells?
I would use 2ul of the ligation mixture.
good luck !!!