TOPO-Transformation Problem - (Dec/04/2009 )

Hi Guys,

I am having problem with using TOPO Vector using as a vector to insert a pcr product of 500bp.

I followed the TOPO cloning Reaction protocol from Invitrogen.

After Transformation,Incubation ,the colonies grew well but I couldn't see any band in almost 20 from 24 colonies(I made patch plate having 24 patches) when i screened through colony pcr.But there was 2 bands around 500 bp size from two colonies from patch plate.I did mini prep with from the bacterial colonies from the patches that I saw bands on.I couldn't see any thing on Agarose Gel after colony PCR.I am using 0.9% agarose Gel.

I am also suspecting LB ,Amp plates.So I streaked Plasmid having Chloramphenicol resistance gene on LB?Amp Plate to check for the quality of LB/Amp plate.Plasmid having CM resistance gene grew on LB/Amp Plate. It also gew on LB,Amp,Glucose plate.(50microgram/mL of Amp and 0.5 % Glucose).So I am confused about the result.So please let me know If there is a way to find out about the quality of LB/Amp,LB/AMp/Glucose plates?Because I am going to use LB/AMp/Glucose plates for my Transformation reaction.Please help me to solve my problem.

I really appreciate any hepfull suggestions

Thanks

JSB

-JSB-

For my TOPO experiencem Usually after transformation, I will incubate, shaking the reaction into a broth (over night) which content 10mg/ml (or any concentration you think fit) ampicilline, and then plate on LB plate the next day.

The reason for me to do so is because sometimes the ampicilline in plate will degraded from time.

Keep us update your result.

Adrian

-adrian kohsf-

adrian kohsf on Dec 7 2009, 04:37 PM said:

Thank for taking time to respond to my question.I was kind of disappointed as I didn't see any responses for my Problem.

Sure,I will update.

Thanks

-JSB-

It sounds like your antibiotic has degraded. Make up a fresh stock and ensure your medium is below 50 deg C before mixing it in.

Cam resistant plasmids shouldn't grow on an Amp plate unless they also have an amp resistance gene.

-bob1-

Not sure that this is the answer, but I've found that colony PCR is unreliable. Generally if it comes up positive, the insert is there, but even if I comes up negative the insert might be there. It's quicker and cheaper than growing them all up and doing mini preps, but not really reliable. Also, I grow things on Amp200 plates. Good luck.

JSB on Dec 4 2009, 09:27 AM said:

-microgirl-