gateway system for cloning - (May/09/2006 )

i was looking for a step by step protocol for the gateway system of cloning using pcr, thanks...viv

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what about reading the manual? is't quite comprehensive

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quite so, but i don't have access to it. i'll check if i can download it from the net, or if you know a url from where it can be downloaded, i should feel grateful to receive it....

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google?

http://www.invitrogen.com/content/sfs/manuals/gatewayman.pdf

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got it, thanks

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I would try using less enzyme than they prescribe.

My lab is currently involved in a high-throughput, cloning and expression project involving gateway and we use about a quarter of the volume of clonase they suggest in the manual.

Clonase is not cheap; unless your project somehow requires it (i.e. high throughput), I might stick to double-digests and ligase.

-Matt

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thanks. this would be the first time i would use the gateway system, if i do...i was thinking of it as an alternative, since i am having this big problem with t/a cloning. the cdna is fine, the transformation goes well, but there is just no insert. i have tried a-tailing after using hi-fidelity taq, and also using native taq. further i have tried two different vectors as well...pgem-teasy of promega, and ptz57r/t of fermentas. following ransformation, i always find the vector, but no insert.

i am hence thinking of doing a blunt end ligation, or use linkers, or the gateway system.....

viv

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[/quote]


thanks. this would be the first time i would use the gateway system, if i do...i was thinking of it as an alternative, since i am having this big problem with t/a cloning. the cdna is fine, the transformation goes well, but there is just no insert. i have tried a-tailing after using hi-fidelity taq, and also using native taq. further i have tried two different vectors as well...pgem-teasy of promega, and ptz57r/t of fermentas. following ransformation, i always find the vector, but no insert.

i am hence thinking of doing a blunt end ligation, or use linkers, or the gateway system.....

viv
[/quote]

Have you tried cutting the PCR product with restriction enzymes for a ligation? I usually do this with a double digest in one step and it works out fine with pET vectors.

-Matt

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[quote name='MisticMatt' date='May 13 2006, 05:35 AM' post='51656']
[/quote]


thanks. this would be the first time i would use the gateway system, if i do...i was thinking of it as an alternative, since i am having this big problem with t/a cloning. the cdna is fine, the transformation goes well, but there is just no insert. i have tried a-tailing after using hi-fidelity taq, and also using native taq. further i have tried two different vectors as well...pgem-teasy of promega, and ptz57r/t of fermentas. following ransformation, i always find the vector, but no insert.

i am hence thinking of doing a blunt end ligation, or use linkers, or the gateway system.....

viv
[/quote]

Have you tried cutting the PCR product with restriction enzymes for a ligation? I usually do this with a double digest in one step and it works out fine with pET vectors.

-Matt
[/quote]

hi matt

no, i haven't. the pcr product is the outcome of primers based on conserved sequences of genes related to the one i am looking for, so no restriction site info is available, and the primers themselves do not carry a restriction site.

viv

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Add a restriction site to the 5' end of the primers. The entire primer does not have to anneal in order for it to amplify (the 3' end is what matters).

Use a rare cutter, like Not I, to reduce the possibility of the restriction enzyme cutting within your insert of unknown sequence.

-Matt

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