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Problem with ligation


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#1 laurequillo

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Posted 10 August 2010 - 10:50 AM

Hi guys, I have a problem doing a ligation and I dont understand the result...

I want to clone my gene from pcdna3 into pbabe. I remove the insert from pcdna3 with EcoRI and XhoI, and I cut the pbabe with ecorI and SalI (compatible with xhoI). I digest 4 ugr, I run a gel, I cut the bands ( the insert is about 3000 bases), purified them, and did the ligation with different ratios of vector:inser, and a negative control, overnight, and then transformation.
I got some colonies in the negative control, but much more in the ligation. I picked up several colonies, I digested them to check that the insert was inside and all of them had the insert. Now the strange part: when I sequence the clones all of them were pcdna3+insert. How come? If I purified my insert, and my vector is pbabe, how I managed to get that result?
I did not do the actual digestions or the ligations, but I provided the plasmids, the enzymes, I saw the picture of the gel, and the bands that were purified, and everything looked ok. (the only thing that occurs to me is that the purified bands were mixed, but the person who did it is quite sure that she did not mixed them...)

Well, let me know what you think, and if you see another explanation (other that the obvious "plasmids or bands were mixed"...)

Thank guys!
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#2 NemomeN007

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Posted 10 August 2010 - 10:55 AM

Hi guys, I have a problem doing a ligation and I dont understand the result...

I want to clone my gene from pcdna3 into pbabe. I remove the insert from pcdna3 with EcoRI and XhoI, and I cut the pbabe with ecorI and SalI (compatible with xhoI). I digest 4 ugr, I run a gel, I cut the bands ( the insert is about 3000 bases), purified them, and did the ligation with different ratios of vector:inser, and a negative control, overnight, and then transformation.
I got some colonies in the negative control, but much more in the ligation. I picked up several colonies, I digested them to check that the insert was inside and all of them had the insert. Now the strange part: when I sequence the clones all of them were pcdna3+insert. How come? If I purified my insert, and my vector is pbabe, how I managed to get that result?
I did not do the actual digestions or the ligations, but I provided the plasmids, the enzymes, I saw the picture of the gel, and the bands that were purified, and everything looked ok. (the only thing that occurs to me is that the purified bands were mixed, but the person who did it is quite sure that she did not mixed them...)

Well, let me know what you think, and if you see another explanation (other that the obvious "plasmids or bands were mixed"...)

Thank guys!



what enzymes did you use to check for insert?

#3 laurequillo

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Posted 10 August 2010 - 11:04 AM

I used ecoRI and AgeI (the last one has one site into my insert and no sites on pbabe or in pcdna3)
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#4 NemomeN007

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Posted 10 August 2010 - 11:23 AM

Unfortunately, either mixed or the insert was contaminated with vector (pcDNA). The enzyme pair that you are using will not differentiate the backbones since the fragment length will be the same in both cases....otherwise, i don't see anything out of the ordinary with you posted....going forward, may want to choose an enzyme pair that can discriminate between both cases...

I know..probably not what you want to hear.... :blink:

#5 pDNA

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Posted 10 August 2010 - 11:33 AM

performing the proper controls often prevents pitfalls, as controls treat the following the same as you do for your ligation reaction:
1) Vector only
2) Insert only
3) Vector only + Ligase
4) just water

This will help safe you time and gives you a felling if it is worth to start screening several colonies.

Regards,
p

#6 laurequillo

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Posted 10 August 2010 - 11:33 AM

Unfortunately, either mixed or the insert was contaminated with vector (pcDNA). The enzyme pair that you are using will not differentiate the backbones since the fragment length will be the same in both cases....otherwise, i don't see anything out of the ordinary with you posted....going forward, may want to choose an enzyme pair that can discriminate between both cases...

I know..probably not what you want to hear.... :blink:


No the thing is that I already sequence the clones, so I know they are in the wrong vector, so I will repeat the ligation, but the result was really weird to me..
"He must be very ignorant for he answers every question he is asked" Voltaire

"This is SPARTA!"

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#7 NemomeN007

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Posted 10 August 2010 - 11:40 AM

Yes, but if your purified insert is contaminated with vector, then you will get the same result again after ligation and transformation...you will preferentially transform your "contaminant" and it will appear to be a successful ligation...perhaps an alternative enzyme set will help you "screen" this out and get correct backbone....

#8 pDNA

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Posted 10 August 2010 - 11:44 AM

therefore you do controls in advance :)

#9 laurequillo

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Posted 10 August 2010 - 11:45 AM

Yeah that is right.
I will just repeat it myself doing all the controls...sometimes is not worthy tobe so lazy, right? ;)

Thanks guys
"He must be very ignorant for he answers every question he is asked" Voltaire

"This is SPARTA!"

"Im the goddamn batman"

#10 pDNA

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Posted 10 August 2010 - 11:47 AM

i used to be lazy too ;)
...but it is not worth the time :)

Good luck and all the best!

Regards,
p

#11 laurequillo

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Posted 10 August 2010 - 12:06 PM

Normally I am not lazy...just when I have to do it again myself! :D

But ok, no complaints, it should be a easy one.

See u guys!
"He must be very ignorant for he answers every question he is asked" Voltaire

"This is SPARTA!"

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#12 NemomeN007

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Posted 10 August 2010 - 03:14 PM

Should be easy....famous last words ;) heheheh




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