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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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  #1  
Old 05-13-2009, 06:35 PM
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Thumbs up problem with TOPO TA cloning....



hi...
i m trying to clone a 1.2 kb product into topo but the problem is that even after getting the pcr product right i m not getting any colonies after transformation?? i m using platinum taq from invitrogen and after that i incubate my pcr product with normal taq for 20' at 72C but no results...??
when i try to amplify my pcr products directly with normal taq with an extension time of 20 mins at 72C to my surprise i dont see any band on gel but when the same is repeated with 10 mins at 72C i get my band ... i do not understand wats the problem....this topoclonig as i hv heard is and efficient but why am i not getting any results...??

plz help....

thanks
nusrat
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Old 05-13-2009, 07:13 PM
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Default Re: problem with TOPO TA cloning....

TOPO cloning is awesome, it's worth the investment during the optimization steps.

Are you doing regular PCR on the plasmid to check for the insert?, if so use the extension time that works whether 5 min, 10 min, 12 min ..... Since your product is rather large I would use a large reaction volume (50microliters to 100 microliters) that way the large product from the early cycles doesn't inhibit further thermocycles from producing (remember the increase in DNA is going to bind Mg++ and diminish buffer capacity as well as use up primers and dNTPs, same with Taq; make sure you have enough to begin with).

When my transformations would fail I found that decreasing the salt fixed the problem ( I was doing electroporation). Increasing the dilution factor of the plasmid by tenfolds usually led to successful transformation.
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Old 05-14-2009, 06:31 AM
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Default Re: problem with TOPO TA cloning....

thanks but i m doing genomic pcr and the inserts are always checked....i think the problem is with 3' a addition may be due to some problem or the other taq is incapable of adding a.

nusrat
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Old 05-19-2009, 04:30 AM
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Default Re: problem with TOPO TA cloning....

with regards to TA cloning, there is a need to cleanup the PCR products before cloning it into the TA plasmid. The PCR buffer may contain NH4, which kills the cells for TA cloning.

However, if DNA cleanup is not done properly, small unwanted DNA fragments may still be cloned into the plasmids with higher efficiencies and give you unwanted sequences after sequencing. This has happened to me before.

Does anyone know if doing a gel purification is better prior to TA cloning or is DNA cleanup good enough?
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Old 05-19-2009, 04:31 AM
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Default Re: problem with TOPO TA cloning....

with regards to TA cloning, there is a need to cleanup the PCR products before cloning it into the TA plasmid. The PCR buffer may contain NH4, which kills the cells for TA cloning.

However, if DNA cleanup is not done properly, small unwanted DNA fragments may still be cloned into the plasmids with higher efficiencies and give you unwanted sequences after sequencing. This has happened to me before.

Does anyone know if doing a gel purification is better prior to TA cloning or is DNA cleanup good enough?
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Old 06-14-2009, 11:30 PM
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Default Re: problem with TOPO TA cloning....

Depends on the specificity of the PCR, though I have found that gel isolation in general can usually save a lot of trouble.

When isolating from gel it is important to let the collumns dry for several minutes after the ethanol/buffer wash to remove all traces of ethanol. Elute in MQ or simply ddH2O. For larger fragments, it helps to prewarm the ddH20 or MQ to about 40-50 deg. C. before applying it to the center of the column and let it sit for 2 minutes or so. Never trust nanodrop! I always check my fragment after gelisolation again on gel.

Last edited by Mathijs; 06-14-2009 at 11:32 PM.
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Old 06-15-2009, 01:47 AM
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Default Re: problem with TOPO TA cloning....

Good idea.

I am intending to use gel extraction next time for any TOPO cloning.
And, I usually do what you do for gel extraction. It works to prewarm the water to about 50deg as it allows the ethanol to evaporate.

However, one thing i have overlooked is the fact that I usually trust nanodrop. I will definitely bear in mind to check my fragment after gel extraction on a gel!!!
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Old 06-15-2009, 01:47 AM
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Default Re: problem with TOPO TA cloning....

Good idea.

I am intending to use gel extraction next time for any TOPO cloning.
And, I usually do what you do for gel extraction. It works to prewarm the water to about 50deg as it allows the ethanol to evaporate.

However, one thing i have overlooked is the fact that I usually trust nanodrop. I will definitely bear in mind to check my fragment after gel extraction on a gel!!!
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Old 09-21-2009, 09:46 AM
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Default Re: problem with TOPO TA cloning....

I hav had a lot of problems with my TOPO cloning!
I also gel-purificate my insert after PCR and then I use the Promega kit.
I obtaing a few colonies (about 15-25), and when I analize it, I find that all of them contain the TOPO vector empty, without my insert!!
I thought that was impossible for the TOPO vector to recircularize alone,!!

Do you have any idea of what I'm doing wrong????
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Old 10-27-2009, 08:21 AM
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Default Re: problem with TOPO TA cloning....

TOPO vector can indeed recircularize spontaneously, esspecially at ambient temperatures. If you have used it for many times and if you kept it at higher temp. than -20 for too long, then this may be the cause for failure of insert ligation. I had the same problems. I tried with fresh vector, same procedure and it worked.
I have some other problems though. I am doing some environmental clone libraries with 16S. I stab clones (TOP10 E.coli transformants) onto 96 well agar plate and I send directly for sequencing. The sequencing service constantly reports low yields of plasmid isolation, often causing 50% loss of sequences per plate! When I checked for plasmid presence in my lab (with the same isolation method), everything is OK. Did anyone have same problems?
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