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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.


5kb gene cloning

5kb gene cloning - Molecular Cloning Forum

5kb gene 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 05-24-2010, 06:48 PM
Pipette Filler
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Default 5kb gene cloning



Dear all,
I am trying to clone a 5kb promoter gene. I've tried many different vector and cloning systems like pDRIVE, GATEWAY, pJET and pBLUESCRIPT. But none of them are working for me. Can anybody suggest me any other easier and efficienct way to do it?
Bests,
DG
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Old 06-01-2010, 08:15 PM
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Default Re: 5kb gene cloning

Hello ^^

Are your cells efficient enough? Most of the systems you mencion there work fine most of the time. Sometimes the problem is just that the cells aren't efficient enough to do the job. Cells as efficient as 1x10^6 cfu/ug DNA are good enough to do the job.

How do you amplify your fragment? Check, because if you're using a polymerase without proofreading activity and a blunt-ended vector, there will be no ligation unless you blunt the PCR product.

Viceversa, if you use a polymerase that doesn't leave the adenine overhangs (like Pfx or Pfu) with a sticky-end vector like pGEM-T, you won't have a ligation with your PCR product.

I'd be cool if you stated exactly what's the problem with your work: there are no transformant cells at the selective plate? Or you just have recircularized vector with no insert of interest in it?

Best Regards ^^!
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Old 06-25-2010, 09:21 AM
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Default Re: 5kb gene cloning

A very common problem here has been loss of purified PCR product, and digested vector (if using that method), on those darned kit mini-clean-up columns, if you follow the protocol that comes with the kits. If one is no longer losing most of one's DNA before even starting the ligation/cloning, this really helps the cloning, and you don't need such super-efficient competent cells. The general tip I woule give it to introduce the DNA to be purified much more slowly onto the column, in the binding buffer. Spin much more slowly at the initial stage, wash twice rather than once and leave the column to air dry for a few minutes at the end (and until you can't smell alcohol any more) before eluting the DNA, and to let the elution buffer soak into the column for a good couple of minutes before eluting. This gives great yields and is still pure enough to clone, ABI sequence, transform etc.
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Old 08-02-2010, 01:06 AM
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Default Re: 5kb gene cloning

You did not mention whether your insert was blunt end or sticky end. 5 kb is not that large to get clones. Did you calf-alkaline phosphatase the cut vector? Did you add a phosphate to your insert (if PCR derived) using t4 kinase? My ligation conditions, eppindorf tube overnight floating in wet ice in the frig. Check you ligation on a gel to see band multiples before proceeding with cloning. If your competent cells are in an eppindorf, heat shock for 90 seconds at 42oC. Grow for 2 hrs 37oC before plating. Hope this helps
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