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Cloning large fragments

Cloning PCR large inserts

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9 replies to this topic

#1 bknm

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Posted 15 May 2013 - 08:23 PM

Hi everyone,

I am about to embark on cloning large fragments (10-20kb). My experience in the past has only been with much smaller inserts so I would really appreciate any help/tips. Will ~20kb be possible??

Thanks,

Bknm

#2 Curtis

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Posted 16 May 2013 - 07:01 PM

We failed to clone 15k in the lab, we asked a company to do it for us. It is kinda difficult, especially if you need to assemble the fragment piece by piece.

#3 DrLeo

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Posted 08 July 2013 - 06:22 PM

Hi Curtis,
When you cloned large fragment, say >10kbp, what method did you use from the PCR step to get the insert to Cloning step? Could you please share some experience with me?
I am so thankful for that.
Best.

#4 Curtis

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Posted 09 July 2013 - 07:59 PM

There are two methods for assembling large fragments; the first method is overlap-extension PCR (OE-PCR, also known as fusion PCR). In this method you need to have 20 something bp identical sequence on the ends of two fragments, which are obviously PCR products. During the second PCR these two fragments bind to each other and make a larger fragment. Check Wikipedia for that. For me, the drawback with this method was that the larger the fragments got, the less PCR product was amplified and this was because of the low concentration of templates. I mean there was no way I could fuse larger than 13 kb. Even with the best purification kits I ended up with low concentration of PCR product and therefore my next PCR didn't work very well.

The second method is sequential assembly by unique RE sites. This is an older method and many papers are published about it. Check Peeters et al. 1999 for the assembly of NDV virus genome.

#5 DrLeo

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Posted 10 July 2013 - 09:00 PM

Thanks Curtis,
Have you ever tried Fusion-cloning with multiple fragments?

#6 Curtis

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Posted 11 July 2013 - 08:18 AM

yes, it's difficult. if your genome is ~10k do it yourself. if larger, ask companies to synthesize and save time.

#7 DrLeo

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Posted 11 July 2013 - 09:47 PM

Hi Curtis,
Maybe you used In-fusion cloning as you said. I tried to perform In-fusion cloning with 3 fragments (each 3-4kbp) into a 4.2kbp vector. What the concentration of each I should use for 10ul reaction volume?
Thank you very much for sharing your experience.

#8 Curtis

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Posted 11 July 2013 - 10:27 PM

add as much as you have, that's the key. low concentration won't fuse.

#9 DrLeo

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Posted 22 July 2013 - 01:10 AM

Thanks Curtis,
But is there any chance to clone 3 fragments of 3kbp of each into a vector?

#10 Curtis

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Posted 22 July 2013 - 07:57 AM

you mean clone sequentially? Yes, of course, but it's not easy, because it depends on the RE sites. It's complicated. Read Peeters et al. 1999 and you'll understand.

OE-PCR is easier, and you will clone in one run rather than 3 rounds.





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