Vector + Insert = almost 12kb
Vector:Insert ratio used; 1:2, 1:3, 1:4
Controls: Vector + Ligase only, Uncut Vector w/o ligase
Ligation: O/N at 16 C
Transformation: About 2ul into DH5alpha electrocompetent cells. Plated all the cells unto LB-Amp plates and incubated O/N at 37 C
Results: No colonies in all plates including controls.
Any ideas what could have gone wrong?
P.S: I've done this experiment before using chemically competent DH5alpha cells with similar colonies. However, there were colonies for the positive control. I switched to electro-competent cells because I thought the size of the construct may be responsible for poor transformation.
Hi there!
Before I comment on the problem, I like to clarify whether you are using the proper vector to insert ratio or not. Is your insert size bigger than your vector? Only then can you justify using more of vector than insert in your ligation reaction. Otherwise, it is generally the other way round (depending on your insert size) i.e. one unit of vector is taken along with 3 or more than 3 units of insert.
Coming to your problem, as you said if you are not getting any colonies in uncut vector transformed plate as well it can have the following possibilities-
1. The vector doesn't have the selection marker which you are using to select your transformants. So even if the transformation is efficient, you are losing the transformants by negatively selecting them and not allowing them to grow. If that is really the case then colonies should appear in plate without any antibiotic. To identify the non transformants which are also going to appear in a plate without any selection pressure, you can plate cells with and without recovery and compare the number of colonies you get. If transformation has happened then more number if colonies are expected after recovery. Although it is not a full proof method, but you may still give it a shot.
2. Your competent cells ain't competent enough. The plasmid size is inversely proportional to the transformation efficiency so in your case (12Kbp) efficiency is expected to be low but not zero! To check that you can use any other vector backbone with a different antibiotic ( which you have used before and has worked) to check if your process of transformation is working and if there is any problem with the DH5alpha you are using.
Hope it helps!
Hi Aderibigbe, then it is absolutely fine. Not getting positive clones can be solved by increasing the insert content and using higher insert to vector ratio, but appearance of no colonies is different and the problem lies somewhere else. I don't think ligation is a problem here as at least vector religation or incomplete digested vector should be able to enter the cell and form colonies on plate. Hope it helps!
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