clonıng problem...

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biolog35
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clonıng problem...

Post by biolog35 » Thu Sep 04, 2008 8:11 am

ı have a problem wıth clonıng.ı dıgested my plasmıd wıth bamHI and ı partıally dıgested genomıc DNA OF bacterıa wıth Sau3A and then ı lıgated fragmets above 1 kb into plasmıd and then ı performed transformation and ı plated transformatıon mıxture on plates ı performed thıs four tımes but there were no colonıes.any ıdea about reasons? ı am tryıng to construct gDNA lıbrary

blcr11
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Post by blcr11 » Thu Sep 04, 2008 3:32 pm

Hard to troubleshoot without seeing everything. Are you certain that the restriction enzymes have been inactivated as well as they can be, either by heat inactivation and/or phenol/CIAA extraction? BamH1 is sometimes a fussy enzyme; are you certain it cut to completion? And are you fairly certain about the concentration of ends within your ligation reaction? It doesn't need to be perfect, but should be within an order of magnitude of target. I'm assuming that your competent bacteria are still viable and competent--that is, they are transfectable with a control plasmid of some kind, and that you know your ligase is still in decent shape.

hilim
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Re: clonıng problem...

Post by hilim » Wed Sep 17, 2008 10:15 pm

as i knoww bamh1 is having star activity- you be carfull- use the apropiate buffer and amount of enzyme :lol:

pguo
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Re: clonıng problem...

Post by pguo » Fri Sep 19, 2008 4:14 am

I think there are some good protocols and troubleshooting you can refer on the topic of molecular cloning over there.
Molecular cloning and more:
http://www.e-biotek.com/index.php?optio ... &Itemid=54

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