trouble with TOPO TA CLONING KIT for SEQUENCING - (Jul/31/2005 )
Hi all,
I have been trying to clone my PCR insert using TOPO TA CLONING KIT for SEQUENCING. I get hundreds of colonies on ampicillin plates when I use my glycerol stocks. According to invitrogen protocol only positive colonies should show up on the amp plates but when I try to cut out my insert from the plasmid (using Eco.R1, that should cut in front and at the back of my insert) , I get only one band on the gel and that too of the size of the plasmid. I do not know what’s going on. Can anyone suggest something?
Everything and anything helps. Thanks in advance.
Which polymerase or polymerase-mixture are you using? Sure it leaves an A overhang?
Apart from that, try blue/white-screening, check your competent cells if you have to induce or not. This way you can cut down on the number of clones you have to check.
How much PCR-product do you add? Too much isn't good for your topo-reaction.
What do you mean with hundreds of colonies when using glycerol stock? You glycerol-stock your PCR-product? or you did topo, grew colonies, made glycerol-stock and afterwards regrew them and checked them for insertion?
Or are they other bacteria and you use these as a control for amp in your plates?
I have been trying to clone my PCR insert using TOPO TA CLONING KIT for SEQUENCING. I get hundreds of colonies on ampicillin plates when I use my glycerol stocks. According to invitrogen protocol only positive colonies should show up on the amp plates but when I try to cut out my insert from the plasmid (using Eco.R1, that should cut in front and at the back of my insert) , I get only one band on the gel and that too of the size of the plasmid. I do not know what’s going on. Can anyone suggest something?
Everything and anything helps. Thanks in advance.
Hi,
I get good number of colonies after I regrow the colonies using glycerol stocks of my cloned E.coli. Since I use the TA kit the Taq polymerase adds the A overhangs. I use 3-4 microlitres of my PCR product.
Thanks
As vairus suggested, I would use the blue/white screening. Plate 40ul of a 40% X-gal solution on your Amp plate prior to plating your cells and only clones that contain your insert will be white.
I use white-blue method as well. And it works very good for me.
Just remember that if your PCR product is very small or in frame you may still get blue colonies on X-Gal plates
Daniel
Longer automated DNA sequencing reads
Hi, I have a long insert (1200bps). Is it possible that the bacteria can kick the insert out?
I've done succesfull topoTA with the same kit up to 3,8 kb, so it shouldn't be a problem unless your gene is toxic to e. coli maybe?
If you have primer dimers in your PCR then you can end up cloning these. Also the 5' nucleotide on your primers affects the efficiency of the 3' A overhang added- you might have chosen a primer(s) that don't add 3'A overhangs efficiently.
Daniel
DNA sequencing analysis software