Ligation problem? - (Apr/11/2008 )
I've been trying to ligate an insert ('round 600 bp long) into a plasmid vector pBluescript, and trasnsform the bacteria afterwards, but something doesn't work, and I suspect it's the ligation step.
Both the plasmid and the insert are cut by the same REs - Eco R1 and Bam H1. Separated by 1% Agarose gel, stained with ethidium bromide, and purified from the gel.
Ligation was mixed with Fermentas T4 DNA Ligase, and I've tried different conditions - 1h on 22°C, overnight on 16°C, overnight on 4°C. Ligation was done over 15 times, with no result. Either no colonies appear after transformation, or the colonies that do appear are negative. I've tried different ratios of the insert vs. vector too. I don't know what to do.
Can someone help me, or give me any suggestion.
Thanx.
Are you treating your vector with alkaline phosphatase to make sure it doesn't ligate back together?
How long did you digest the vector and insert containing plasmid?
Try shorter digestion times like 1 hr.
Is your insert a PCR product?
I haven't tried that. But it shouldn't ligate back together since it was cut by 2 different RE. The sticky ends are not complementary.
Try shorter digestion times like 1 hr.
Is your insert a PCR product?
Digestion was done for 1-2h. The insert was cut by double digestion, with both enzymes together. And the vector was at first also cut by double digestion, but later on I tried to digest it with one ezyme at the time (I thought maybe the restriction sites are too close for double digestion - they are around 10 bp away).
And the insert is not a PCR product, it was cut from another plasmid vector.
The vector should not self-ligate as the restriction sites are different.
Is your insert a PCR product?
I haven't tried that. But it shouldn't ligate back together since it was cut by 2 different RE. The sticky ends are not complementary.
True
I should have read your post properly 
HAPPY FRIDAY! wOOt!
Can you try with a new ligase and buffer.
how much DNA do you use for ligation?
Try shorter digestion times like 1 hr.
Is your insert a PCR product?
Digestion was done for 1-2h. The insert was cut by double digestion, with both enzymes together. And the vector was at first also cut by double digestion, but later on I tried to digest it with one ezyme at the time (I thought maybe the restriction sites are too close for double digestion - they are around 10 bp away).
And the insert is not a PCR product, it was cut from another plasmid vector.
1-Can you sequence your plasmid from which you are cutting your fragment to make sure that there is not more sites than you think.
2-can you try different ligase+buffer
3-as a control, do you have any other cut plasmid with the same enz that you can try and the other way a fragment with the same end to try with your pBL
4- just to keep in mind for future ligations,dephosphorylation of the vector help even if you have different sites in case of one enzyme cut and not the other( a gel can tell you that your plasmid is linearized but will not tell you that both enzymes did cut). It help lower the background .
good luck