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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.


TOPO TA cloning

TOPO TA cloning - Molecular Cloning Forum

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 09-16-2008, 09:20 AM
Pipette Filler
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Unhappy TOPO TA cloning



Hi,

I have a problem to clone a PCR product into the pCR2.1 TOPO vector. The PCR product (300 bp) was synthesised with the Platinum taq DNA polymerase Hifi from Invitrogen. After the gel electrophoresis, the product was extracted with the Qiaex geletraction kit.


For the TOPO cloning, I used the following mix:
(TOPO TA cloning KIT with pCR 2.1-TOPO from Invitrogen)

purified PCR product: 1ul to 4ul (between 10ng and 100ng)
TOPO vector: 1ul
Salt solution: 1ul
add bidest water to 6ul

After 30 minutes of incubation at RT, I used 2ul to 4ul for the transformation into the TOP10 E.coli cells.

Result: A reduced number of clones (90 clones) where 2/3 of the clones were blue


I checked the transformation efficiency of the cells: OK
I made an A-tailing after gel extraction: no change

What I am doing wrong?
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Old 02-06-2009, 01:39 PM
Pipette Filler
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Default Re: TOPO TA cloning

I don't understand what is wrong? You got white colonies didn't you? So you have some positive clones?
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