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Cloning advice needed


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4 replies to this topic

#1 FlyerGirl

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Posted 09 February 2010 - 05:24 AM

Hi

I am new to cloning and need to clone 5 genes of interest into mammalian expression vectors. 4 of the 5 have been done with no problems but the fifth is turning into a bit of a problem. I have been trying to clone it for several months now and would appreciate any suggestions for things I may be doing wrong or should try.

I have the cDNA I am interested in, in a TOPO vector. I have been trying for several months now to get it from here into a mammalian expression vector.

I have used the plasmid in a PCR to amplify the insert with different restriction sites on both ends (EcoRI and NheI). I get a good yield from the PCR reaction. I have then tried to ligate this into both pcDNA3 and pEF (a pcDNA3 derivative with an elongation factor promoter rather than the CMV promoter). I have tried transforming the ligation reaction into both DH5alphas and TOP10F, I get plenty of colonies but when I miniprep and restriction digest them I get nothing but empty vector.

I have also tried restriction digesting the insert out of the TOPO vector and ligating it into pEF. In this case the insert has the same restriction site on both ends (EcoRI). When I analyse the colonies I get, about half of them have the insert, but in all I have tested the insert is in the wrong orientation.

This makes me wonder if I am getting some kind of expression of my insert in the bugs (is this even possible from a mammalian expression vector?) and it is toxic.

Any thoughts/suggestions on what the problem might be or any alternative methods that are worth trying would be really appreciated.


Many Thanks

FlyerGirl

#2 HomeBrew

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Posted 09 February 2010 - 06:16 AM

I suspect you have one of two problems -- either the NheI is not cutting, or the gene is toxic to your recipient cells.

How many 5' bp have you added to the NheI site on your primer?

#3 snoopyx

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Posted 09 February 2010 - 09:19 AM

When I was trying to clone a glycoprotein all my minis showed a very strange pattern after a control digest. None had the correct bands. The solution was pretty simple. It was toxic for the bacteria so the insert got partially kicked out. Try to use low-copy vector and clone it in there.

Edited by snoopyx, 09 February 2010 - 09:19 AM.


#4 FlyerGirl

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Posted 10 February 2010 - 10:22 AM

Thanks for your replies.

I have 6 bp 5' of the NheI site. I wondered about whether this enzyme was cutting (I have no experience of using it previously) so am going to order the same primer but with a different restriction site encoded to see if this solves the problem.

I will also ask around the lab to see what other vectors I can try- I'm not sure if anyone uses a low-copy number one. If it turns out that the gene product is toxic are there any vectors from which the insert won't be expressed in bacteria?

Many Thanks

#5 phage434

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Posted 10 February 2010 - 04:05 PM

Lucigen markets a set of vectors with transcriptional terminators surrounding the cloning site, to eliminate accidental transcription.

You might also want to check the sequence to look for cryptic bacterial promoters within the coding region.




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