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Insertion of fragment with same 5' and 3' ends


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#1 flygirl

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Posted 08 January 2010 - 02:44 AM

Hi all,
I have been trying to make a construct by restriction cloning that requires insertion of a fragment that has EcoRI sites on both 5' and 3' ends. Before setting of, I estimated that statistically there would be a 50:50 chance that fragment would be inserted in either correct or reverse orientation. This turns out not to be the case at all, with the correct orientation only occurring in 9 out of 140 colonies.

Am now wondering what decides this process...

#2 phage434

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Posted 08 January 2010 - 05:45 AM

Possible selection due to slower growth of the cells containing the insert in the "correct" orientation.

#3 FlyerGirl

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Posted 11 January 2010 - 05:36 AM

I am having the same problem as flygirl, except in my case I have yet to get any clones with the insert in the 'correct' orientation but I have plenty in the 'reverse' orientation. It has been suggested to me that the insert may be toxic to bacteria, but as I am cloning into a mammalian expression vector (pcDNA3) should this be a problem? If so is there any way around this?

Many Thanks

#4 dpo

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Posted 11 January 2010 - 05:54 AM

from http://tools.invitro...sionVectors.pdf

"Cryptic E. coli promoter - likely to be somewhere in the CMV promoter. When the gene of interest is in the correct orientation for eukaryotic expression, the cryptic E. coli promoter will often express the gene in E. coli. The level of toxicity to E. coli will depend upon the particular gene product and upon the efficiency of translation initiation. One way of getting around this problem is by engineering a prokaryotic transcription terminator , upstream of the gene of interest. This will result in reduced expression in E. coli without altering eukaryotic expression."

So your GOI might be expressed in your bacteria and might be toxic there.

#5 flygirl

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Posted 15 January 2010 - 04:57 AM

Thanks for the replies!

I had been wondering about the possibility that the E.coli are able to express my protein of interest. Even at low levels it might be toxic.... We have the gene of interest in a mammalian (CMV promoter) and we've found that mammalian cells transfected with this construct are very sick (bleb, die). Bugs transformed with this construct do not grow very well either; I tend to grow minipreps for much shorter than usual (6hrs) or at lower temperature (25-27C) and then manage. At 37C anything that grows has the construct, but horribly "scrambled".

Was a bit surprised to find that now that I am trying to subclone this gene into a fly expression vector (under pUASt promoter) I am getting the same problem. Have now managed to get my construct, just by sticking my head down and just doing tons of colony PCRs.

This might be a way to go for you as well flyergirl:
1. Set up your ligation and grow your plates ON at RT (not in 37C)
2. Then pick colonies onto a grid (agar plate that you grow again at RT), and into a PCR mix (can do 96-well plate). For primers you should pick one of the pair in your vector, one in your insert.
3. Run gel to see if you've got a band.

Fingers crossed.

#6 flygirl

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Posted 15 January 2010 - 04:58 AM

in addition.
NEB (and also Invitrogen and Statagene) do E.Coli that can handle toxic DNA better. I have no experience using these, but maybe some one else has..?




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