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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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TOPO TA cloning
Posted by: gouri chatterjee (IP Hidden, New member, 1)
Date: March 10, 2008 10:52AM
Hi
I have been trying to clone a 6KB PCR product into TA cloning vector, but only a portion of the fragment is getting cloned. I ran the PCR product and also RE digested TA vector with supposedly containing the insert, and there is a size difference in them.I sequenced and found about 800 bases are missing . Could anybody has some suggestions for me. Thanks gc
Re: TOPO TA cloning
Posted by: drsgarrett (IP Hidden, New member, 3)
Date: April 4, 2008 03:33PM
I've recently cloned both a 4kb and a 5kb fragment into Topo as well as pGL vectors.
First, are you gel purifying your PCR products? If not, the issue is that you have multiple products from early termination and such, and the TOPO is preferring the smaller ones. Do enough PCR so you can do several lanes so you'll get back enough of the product to do the transformation with. Second, use all of your cloning reaction mixture in one of your transformation conditions. Leave the cloning reaction on the bench for the longest time suggested by the book (up to 20 minutes I think), then put into your compentent cells and incubate on ice the full 30 minutes. This maximizes the efficiency in my experience. After that, screen, screen, and screen. If you are only missin 800 bp, odds are, there are reactions with the full amount. Doing the gel purification so that all your potential products are about the same size will maximize your number of positives. sg
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