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Ligation of 16kb PCR product


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#1 Sank

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Posted 10 November 2011 - 01:52 PM

Hi everyone
I have been trying to clone a 16kb fragment into pGEM-T easy vector (3 kb).
So far I have tried 1:1, 1:2, 1:3 and 1:5 vector:insert ratios.
Ligation was tried at 4C, 16C and RT. So far none of these conditions have helped.
After doing the blue white colony screening, I get may be 2 or 3 white colonies, which dont have an insert. The rest of the colonies were all blue.

Meanwhile, I have also been trying to clone the 16kb fragment into pGL3- Basic vector (4.8 kb).
I have used in-fusion kit for this purpose. My primers have HindIII sites and a 17 bp overlap with the vector sequence. I have tried 1:2 vector:insert ratio and incubated the cloning reaction for 15 min at 50C. Empty vector shows lots of colonies. The plate with 1:2 cloning reaction shows 20-30 colonies. None of them have the insert.


Can anyone help me with this? I want to 1st save the insert by putting it into a TA vector. So cloning into pGL3 Basic comes later.
Any suggestions will be greatly appreciated.

Thanks
Sankella

#2 Curtis

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Posted 11 November 2011 - 08:01 AM

Do you realize pGEM cannot easily hold 16 kb?

#3 pDNA

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Posted 11 November 2011 - 09:33 AM

have you checked self-ligation properties of insert and vector?
...so that you can rule out one of your parts is not properly digested.
I would do that first before taking any other measures ...because maybe it has nothing to do with insert size.

If you come to the conclusion that it is due to the insert size than i would go for the Takara Ligation Kit Long ...maybe this one can do the trick!

Best regards,
p

#4 pDNA

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Posted 11 November 2011 - 09:46 AM

i don't know for what reason you do that cloning
...but maybe it is also an option to use another plasmid backbone like pBeloBac11 since as far as i know pGEM is a high copy vector.

What kind of DNA are you cloning? ...eukaryotic or prokaryotic sequence?

Regards,
p

#5 Sank

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Posted 11 November 2011 - 10:15 AM

Thanks Curtis and pDNA

I spoke to the company about pGEM-T and they said it worked for 20 kb.

I am trying to clone a 16kb promoter of a eukaryotic gene.
How do I check if the insert is self ligated?

I will think about the Takara kit, may be that will help.


Regards
S

#6 pDNA

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Posted 11 November 2011 - 10:35 AM

you can check that easily by setting up a ligation reaction with solely the insert (~50 ng ...so that you can see it on an agarose gel) and ligase.

If both restriction sites are digested properly you should get multiple bands (32 kb, 64 kb, and so on ...should look like a ladder) ...due to the large size you'll have to use a low percentage agarose gel ...to see the multimerization bands.

The same can be performed for the digested vector as well.

Regards,
p




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