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Insert much smaller than expected after miniprep


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#1 Firenzi

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Posted 04 December 2010 - 08:55 AM

Dear Colleagues,

Ive been working on a bacterial transformation for almost one year, with no results or strange ones.

Here are the facts. I received a plasmid (pUC19 + insert of ~10kb) fixed on filter paper. I eluted it using TE buffer and used it (1ng/uL) for heat-shock transformation on DH5a cells.

After plating transformed bacteria on a selective plate (agar + 100ug/ul Ampiciline), I could find only one or two colonies. An initial screening (PCR: M13F + Insert reversed primer) confirmed that the plasmid are present on those colonies. Other PCRs to different regions of insert confirmed that the insert are on his full-length on those colonies.

Then, I inoculated those colonies on 3 mL of LB (100ug/ul AMP) and incubated O/N at 37C. The cells grew nice. I purified it using fermentas miniprep and did a XbaI digestion to linearize the plasmid and ran it on a 0,8% agarosis.
Surprisingly, the linearized plasmid has almost HALF (6kb) the size of the original one used to transform the bacteria. I tested this smaller construct and it seems that the middle of the insert has gone. I can amplify only the first 1500nt on 5 region and the last 1500nt on 3 part. Where is the rest of my insert? Is this a recombination event? Deletion? Is there anyway to prevent it?

I repeated the transformation almost 10 times and used several approaches (grow at 30C, glucose rich medium, increase and reduce amp concentration, different heat shock conditions and electroporation ) and obtained the same result, a construction with half of expected size.

Does anyone can give me any light on this problem? I have no more ideas.

Best regards,

Firenzi

#2 GeneTurk

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Posted 04 December 2010 - 09:41 AM

Hello Firenzi,
I think you are having star activity with your XbaI enzyme. please check if there are any similar regions to the cutting site of XbaI. Have a look at this NEB link for star actvity. I hope this helps to resolve your issue.
http://www.neb.com/n...ar_activity.asp

#3 phage434

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Posted 04 December 2010 - 09:46 AM

Are you certain the original plasmid really was full length? Can you PCR amplify the internal regions from your original source? Sequence it? Have you tried cloning into a different strain, such as SURE or STBL2?

#4 Firenzi

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Posted 04 December 2010 - 11:41 AM

Hello Firenzi,
I think you are having star activity with your XbaI enzyme. please check if there are any similar regions to the cutting site of XbaI. Have a look at this NEB link for star actvity. I hope this helps to resolve your issue.
http://www.neb.com/n...ar_activity.asp


Geneturk,
If I'm having a star activity i would expect two or more bands on agarose gel, right? After miniprep digestion I can see only a single sharp band with 6 kb. So I think in our case, Star activity is not the problem.

Are you certain the original plasmid really was full length? Can you PCR amplify the internal regions from your original source? Sequence it? Have you tried cloning into a different strain, such as SURE or STBL2?


Phage434,

I'm certain that the original plasmid is in its full length, I've amplified several internal regions on the source. Clean nice sharp bands. Also tested with M13 primers to confirm that insert is ligated to plasmid.

Also I have not tested cloning on those strains since we don't have it on our lab. Moreover, a few other researchers around the world succeeded clone this plasmid on DH5a strain. I've followed strictly their protocols with no success. We have BL21 strain, should I try clone on it?

#5 Firenzi

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Posted 07 December 2010 - 02:49 AM

C'mon guys...

Any ideas??

#6 phage434

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Posted 07 December 2010 - 07:42 PM

BL21 would be a very bad choice of strain to attempt cloning with. Your choices boil down to finding and using a more stable cloning strain, or finding out more about what exactly is happening. Do you have sequence for the construct you want? Do you have sequence for the plasmid that you created? That would be my first order of business. Then, I would look at what repeats and homologies exist that might explain the recombination you are apparently seeing. You'll know a lot more after doing this, if only to know that you have a severe problem. More likely, you will discover the problem is something entirely else that we haven't thought of. You should be collecting all of the information you can, using whatever tools are available, to figure out what is happening. Have you done restriction digests of the plasmids you create? These are simple and inexpensive. You can't figure out what is wrong by asking others or by waiting.

#7 perneseblue

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Posted 11 December 2010 - 12:44 PM

Much that I would say has already been stated by phage434.

However I would add that something isn't right about the initial transformation

After plating transformed bacteria on a selective plate (agar + 100ug/ul Ampiciline), I could find only one or two colonies.


You should have gotten hundreds of colonies. Getting 1-2 colonies is wrong. Something isn't right with either the cells, the plasmid or type selection agent. You might want to keep your mind open to the idea that the plasmid was not construct correctly to begin with.
May your PCR products be long, your protocols short and your boss on holiday




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