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Has anyone used In-Fusion Cloning Kit? - (Nov/29/2005 )

Hi all,

I am trying to clone my PCR product (flanked with 15 bp of homology to the vector ends) to my own vector (not the one BD provides with the kit). I have carefully digested my vector and purified it from the gel. I have also purified my PCR product using differend methods such as column purification, gel purification and gel purification followed with a phenol:chloroform extraction. After the recombination reaction nothing really seem to work. My positive control reaction results in about 100 colonies but my negative control reaction and real reaction seems always the same (only few wrong colonies). Has anyone used this kit? I'd really appreciate any hints or tip from other users. Or is it just so that what the kit promises is too good to be true sad.gif

Best,
LentiMan

-LentiMan-

Hi

I used the in-fusion kit and it worked really well - i'm not sure what could be the cause of your problem.

I used a different plasmid to that in the kit, and cut it with 2 different restriction enzymes. (as recommended). I didnt CIAP the vector as the ends were non-homologous. I cleaned up the PCR using a gel extraction, mixed it with the plasmid in the In-fusion reaction and hey presto, lots of clones, all containing the insert in the correct orientation.

I can only suggest that you have designed the homology to the plasmid incorrectly - is the forward primer sequence homology the same as the plasmid, and the reverse primer sequence the reverse complement of the plasmid? I nearly ordered the wrong primers myself!!

Maybe worht getting someone else to check as anyone can get it wrong!
Frustrated.

-frustrated-

QUOTE (frustrated @ Dec 10 2005, 04:45 PM)
Hi

I used the in-fusion kit and it worked really well - i'm not sure what could be the cause of your problem.

I used a different plasmid to that in the kit, and cut it with 2 different restriction enzymes. (as recommended). I didnt CIAP the vector as the ends were non-homologous. I cleaned up the PCR using a gel extraction, mixed it with the plasmid in the In-fusion reaction and hey presto, lots of clones, all containing the insert in the correct orientation.

I can only suggest that you have designed the homology to the plasmid incorrectly - is the forward primer sequence homology the same as the plasmid, and the reverse primer sequence the reverse complement of the plasmid? I nearly ordered the wrong primers myself!!

Maybe worht getting someone else to check as anyone can get it wrong!
Frustrated.


I read a paper "Comprehensive analysis of transcriptional promoter structure and function in 1% of the human genome". In the Method part, they used InFusion cloning system (BD Biosciences) to clone PCR products into pGL3 baisc luciferase vector (Promega). I also used the same kit to clone my PCR product to pGL3 promoter luciferase vector (Promega). However, I only obtain very few clones(3 ~4). I used the same restriction sites (MluI and BgllI) as they designed in that paper for cloning reaction. Because there are only 3 nucleotides different between pGL3promoter and pGL3basic, I changed the first 3 nucleotides in my reverse primer, but it doesn't work well. The 15 extend primers is as follow as: Please give me some suggestions to solve my painful problem. I really appreciate, thanks



Forward:

5' ccgagctcttacgcgt-my own sequence 3'



Reverse:

5' tgcagatcgcagatct- my own sequence 3'


Rreverse primer of that paper is:


5' cttagatcgcagatct- my own sequence 3'
(only first 3 nucleotides are different as mine)


ps: I purify vector and PCR product by QIAGEN gel extraction kit, and the primer ordered from Invitrogen

Best Regards

-albertchen1-

QUOTE (albertchen1 @ Jul 22 2006, 01:25 AM)
QUOTE (frustrated @ Dec 10 2005, 04:45 PM)
Hi

I used the in-fusion kit and it worked really well - i'm not sure what could be the cause of your problem.

I used a different plasmid to that in the kit, and cut it with 2 different restriction enzymes. (as recommended). I didnt CIAP the vector as the ends were non-homologous. I cleaned up the PCR using a gel extraction, mixed it with the plasmid in the In-fusion reaction and hey presto, lots of clones, all containing the insert in the correct orientation.

I can only suggest that you have designed the homology to the plasmid incorrectly - is the forward primer sequence homology the same as the plasmid, and the reverse primer sequence the reverse complement of the plasmid? I nearly ordered the wrong primers myself!!

Maybe worht getting someone else to check as anyone can get it wrong!
Frustrated.


I read a paper "Comprehensive analysis of transcriptional promoter structure and function in 1% of the human genome". In the Method part, they used InFusion cloning system (BD Biosciences) to clone PCR products into pGL3 baisc luciferase vector (Promega). I also used the same kit to clone my PCR product to pGL3 promoter luciferase vector (Promega). However, I only obtain very few clones(3 ~4). I used the same restriction sites (MluI and BgllI) as they designed in that paper for cloning reaction. Because there are only 3 nucleotides different between pGL3promoter and pGL3basic, I changed the first 3 nucleotides in my reverse primer, but it doesn't work well. The 15 extend primers is as follow as: Please give me some suggestions to solve my painful problem. I really appreciate, thanks



Forward:

5' ccgagctcttacgcgt-my own sequence 3'



Reverse:

5' tgcagatcgcagatct- my own sequence 3'


Rreverse primer of that paper is:


5' cttagatcgcagatct- my own sequence 3'
(only first 3 nucleotides are different as mine)


ps: I purify vector and PCR product by QIAGEN gel extraction kit, and the primer ordered from Invitrogen

Best Regards



I'm using the in-fusion method and have reduced number of colonies. Does anybody know if incomplete digestion by 2 different restriction enzymes may affect the in-fusion reaction i.e the presence of the uncut vector.

-mwwood-