Please Help me

1 post / 0 new
protein's picture
Please Help me

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