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Molecular Cloning Forum DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


problem with TOPO TA cloning....

problem with TOPO TA cloning.... - Molecular Cloning Forum

problem with TOPO TA cloning.... - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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  #31  
Old 05-22-2010, 05:44 AM
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Default Re: problem with TOPO TA cloning....

yea..
that's actually what I meant..
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  #32  
Old 12-05-2011, 04:19 PM
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Default Re: problem with TOPO TA cloning....

Quote:
Originally Posted by clevermizo View Post
OK Here's the deal:


According to Invitrogen tech support, 1 uL of TOPO-TA vector contains 10 ng of DNA. Furthermore, the best molar ratio of PCR Product: TOPO vector is actually 1:1. I always do a calculation first and see how many ng of PCR product I need to use in the TOPO reaction, and I get almost entirely white colonies after blue/white selection.

Here's what I do:

Do PCR. If PCR is clean, just do PCR cleanup (Qiagen) to get rid of salts/dNTPs/primers, etc. If it's dirty I do gel extraction. I measure DNA concentration by A260 or run it out on a gel if it's too low.

Then before I do anything else I calculate: (10)*(kb PCR)/(3.9 kb TOPO) = __ng PCR product for reaction. Once I know that I scale up appropriately to a 10-25 reaction volume for adding 3'-A overhangs with Taq (I always use high fidelity polymerases for cloning). A simple 10 min @ 72C always does the trick.

If I need x ng PCR product for TOPO reaction, I plan my 3'A reaction to contain x ng/uL PCR product. This way, I add exactly 1 uL and it simplifies my life. I mean, you do whatever you want, but I highly recommend calculating how much PCR product you need to add to the TOPO reaction before doing it, it really helps out. I always transform into regular, cheap DH5alpha. No need for anything fancy (although Invitrogen does force the TOP10 on you).

Using too much PCR product in the TOPO kit is a bad idea. I've had to dilute PCR product 100-fold. It's not going to disappear. Less is definitely better than more. Too much PCR product will pretty much ensure you have all incorrect clones.
Hi!

I realize this thread is long dead but I want to give this a try nevertheless!

I have just cloned for the first time and obtained plenty of clones, but after PCR screening it appears that my bands give a fragment shorter than the gene I wanted to clone!
I added 4 microlitre of PCR product to 1 microlitre of vector. The reason I did this is because I cut a specific band in a gel and purified it, leaving me with (so I thought) not very much DNA, so I opted to add the maximum amount possible for transformation.
In one of your posts you said that "Too much PCR product will pretty much ensure you have all incorrect clones". Could you please elaborate on this? Is it possible that only part of my gene was inserted into the vector? I am still a bit puzzled why all my obtained fragments are only 500 bases long when I added a purified 1000 base gene to the vector. I am hoping that the incorrect Ratio of vector/ PCR product could be the problem?

Thank you very much in advance
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  #33  
Old 01-15-2012, 12:09 PM
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Default Re: problem with TOPO TA cloning....

I don't think it had anything to do with the ratio. I also always use 4 ÁL PCR product, and especially when it's from a gel extraction I think it sounds like the right amount. I also have the experience that you lose A LOT of material in gel extractions so you need to use as much of it as possilbe in a TOPO-reaction. In my experience the best is if you can get a PCR that is really clean (one single band only) so that you can use it directly and avoid the gel extraction. I would also run some of the gel extraction on a new gel to verify that you didn't lose everything (don't measure the concentration on a Nanodrop, it often gives incorrect readings on gel extractions because of particles from the column).

I have no good explanation why you get a smaller fragment, it seems very unlikely that the DNA would be broken. How did you analyze it? If it was by restriction digestion, are you sure that your enzyme doesn't cut in the middle of the sequence leaving you with two 500 bp fragments?
You could also try to do a PCR on the clones with your original primers and see if you get a fragment what size it is.
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  #34  
Old 01-27-2012, 10:21 AM
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Default Re: problem with TOPO TA cloning....

Hi thank you for your reply!I did try with both the original primers and the M13 primers that come with the TOPO kit. Regarding the ratio, I tried a 1:1 ratio and a 3:1 ratio and I found that actually the 1:1 ratio produces more colonies.
I did try again before Christmas and I sequenced the smaller fragments that I got! They are actually empty vectors! I didn't consider this before as with this kit, everything that grows should contain the insert (or so I understood it)! I tried again this week. I am cloning PCR products, so this time I extended the 'Extension step' by half an hour to make sure I have plenty of A overhangs. It did seem to work fine, I had more than twice as many colonies than before Christmas!
I picked the colonies, and re-peated the PCR with the M13 primers that come with the kit, but again I can see empty vectors in the gel! I am not sure why this is happening. My first thought was that possibly something is wrong with the ampicillin I am using. But somebody else in the lab used the same stock and had no problems at all...
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  #35  
Old 05-08-2013, 09:28 PM
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Default Re: problem with TOPO TA cloning....

Hey Catfish,

Have you tried Blue/White screening your colonies? You'll always have colonies in which the vector re circularized itself and avoided the cell death gene on the topo plasmid. If you blue/white screen, more often than not you'll get colonies with the insert in it as it becomes easier to tell which colonies have at least some sort of insert in it.

Just a note with topo ta, you don't need IPTG to induce B-gal transcription as the plasmid has an always-on inducer gene, so you just need X-gal for the source. Hope this helps!
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