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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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TOPO TA cloning problem
Posted by: onewinged (IP Hidden, New member, 3)
Date: November 20, 2007 02:36AM
Hi all,
I'm facing a bit of a problem with my cloning, and I wonder if anyone can help me. I'm using Invitrogen's TOPO TA kit to clone my insert, but when I first tried it, the reaction produced no colonies. So I did the control reactions according to the protocol: PCR'd the control template, cloned it into TOPO and also did the vector-only reaction. The result: still nothing. Not a single colony, blue or white. Even the vector only-plates should include a few colonies which are resulted in blunt-end ligation, but there was nothing at all! Then I thought my cells were dead (Chem. competent TOP10 cells), so to check this I plated some on just LB, no antibiotic screening. The plates were empty the next day. At this point I decided to do one more test, and transformed the cells with the control plasmid pUC19, which includes Amp resistence gene, and plated the cells on LB+Amp. The result: 87 white colonies. So, what's going on? Is my TOPO vector busted? If so, how would you guys explain that the non-transformed cells wouldn't grow on LB? I'm confused... and thankful for any suggestions, feel free to ask me the stupid questions -there might be a lot of things I haven't concidered.
Re: TOPO TA cloning problem
Posted by: onewinged (IP Hidden, New member, 3)
Date: December 5, 2007 08:31AM
Ok I might have solved something. Seems like my gel purification is where my insert gets lost. I just checked my gel purification product (was using Qiagen's gel purification kit) and there's nothing on gel. Bummer.
Re: TOPO TA cloning problem
Posted by: drsgarrett (IP Hidden, New member, 3)
Date: April 4, 2008 03:38PM
Which gel purification kit are you using? I find the Qiaex gives crappy yields always. Quiagen's column purification works much better. Are you eluting in the given elution buffer or water? If your water's pH is wrong, you won't get anything off the column. In my experience, the EB buffer does not give problems in most downstream reaction.s
Re: TOPO TA cloning problem
Posted by: elm3r (IP Hidden, New member, 3)
Date: April 11, 2008 08:28AM
if you want to purify 2ug of DNA from a gel slice on a Quiagen colum, you are going to end up with 200ng, my own experience ! Always use the TE buffer to elute and start with at least 5 times more DNA than you need for downstream steps...
about the cloning, this TOPO TA cloning thing is funny, it's a complete mess and a joke, it actually mixes the TOPO system found in some Gateway entry vector, so that you can clone directionally your PCR products with the help of one base-pairing site and Topoisomerases... The TA cloning is classical cloning with T overhangs, meaning that you can ONLY clone PCR products that have been generated with Taq-related polymerases... (they add A overhangs to the PCR product)... This is where TOPO TA sucks : you need to have A overhangs on your PCR product otherwise it won't fit in the vector, it's just to "simplify" of the user by making him pay much more money. anyway, as I like to say : The only thing that Eppendorf do well is their Eppendorf Tubes, and 30% of the money going in research actually goes to Invitrogen... thiefs ! :P
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