I have tried cloning my gene into an empty vector and grow them up using E.coli competent cells and have no colonies. I also attempted to clone the gene fragment first using the TOPO TA kit and the Zero Blunt kit and not a single colony. I'm not sure what's going on. Any help will be greatly appreciated.
I am hoping to determine ways to examine the toxicity of the insert sequence.. perhaps using blast.
Dear Valencia
just a question: which vector (and expression under which promoter) and which E.coli cells?
Vector: PET01
I'm not sure what you mean by expression and which protomor...??
E.coli cells: OneShot TOP10 E. coli competent cells
Its not about the toxicity, it is just a compatibility and skill issue.
Most often cloning does not work due to improper and unskilled hands doing the process (personal experience). Additionally, TOPO kits were not the best kits I have used either.
However, is your gene fragment pure and fresh?
And I did not really understand why you are using TA cloning kit and blunt end cloning kit for the same gene fragment? And the name itself indicates, both kits are for different kind of gene fragments? Which one is yours?
How do you screen your clone colonies? Do you use blue-white screening?
How do you plate your transformed cells? How do you incubate your plates? and for how long?
Think about these factors before thinking/calling your gene "toxic".
Hi:
if you use taq polymerase amplified your gene, you could use TA vector, if you use pfu/Q5/phusion polymerase amplified your gene, you could use blunt vector.
do you have a positive control plasmid for transformation efficiency?
Junjie Shao. Yes. I used taq and the blunt end vector. I also tested for a positive control using a cut and uncut vector to test for transformation efficiency and I got hundreds of cells.
abhijeet, i agree with you. I believe it may be technical and potentially a learning curve. However, using a positive control and getting hundreds of cells post transformation leads me to believe that there may not be an issue with the transformation. i will attempt again with supervision from my PI.
UPDATE:
I ran a test digest again and ran it on a gel with the restriction enzymes alone and together. I found that for some reason one of the restriction enzymes aren't cutting at all while the other restriction enzyme cuts effectively but causes linerization. This could be why there aren't any colonies. I have ordered new restriction enzymes.
Nice to see the UPDATES to a question. Appreciated.
However, did you do an in-silico restriction digestion of you fragment with restriction enzymes?
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