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cloning without phosphorylation


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7 replies to this topic

#1 Nayeem991

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Posted 10 July 2012 - 07:52 PM

HI all, I was trying to clone a blunt ended PCR product in pUC19 at smaI site. I don't have PNK right at this moment and it would take few days to get in hand. But cloning this PCR product is an emergency. Is there any way to clone PCR product in pUC without phosphorylating the PCR product? I dont have phosphorylated primers as well.

Edited by Nayeem991, 10 July 2012 - 07:53 PM.


#2 Curtis

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Posted 10 July 2012 - 10:41 PM

I'm afraid there is no other way. Maybe you can look around your lab and ask if anybody has pJET to give you. synthesizing phospho-primers is faster than buying PNK. Can you order those?

#3 Papaver

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Posted 11 July 2012 - 03:41 AM

Well...I know in theory that you need to phosphorylate...but if it's urgent just try without pohosphorylation and repeat at the same time, if you don't have enough material, the cloning so you would be ready when your PNK has been delivered. I once cloned blunt end without phosphylating and I got at least one clone which was enough for me.
Otherwise ask other lab(s) if they can give you a bit for the moment...

#4 phage434

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Posted 11 July 2012 - 11:47 AM

If you use a large excess of insert and low concentration, you may be able to clone by not doing the dephosphorylation of your cut vector. High insert concentration will favor insert vs. religation, and low concentration will favor circularization rather than intra-molecular reactions. Try 10:1 insert to vector and 5 ng/ul final vector concentration. I'd try using quick ligase buffer for blunt cloning.
Be prepared to sort through large numbers of vector only colonies.

#5 Nayeem991

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Posted 11 July 2012 - 08:59 PM

Thanks to all for your suggestions. I was trying to clone the insert without phosphorylation for last few days and luckily got my desired colony today.. Posted Image

#6 Curtis

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Posted 11 July 2012 - 11:28 PM

wow great news, but how is this possible when there is no phosphate group? I'm confused!

#7 Nayeem991

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Posted 12 July 2012 - 03:03 AM

wow great news, but how is this possible when there is no phosphate group? I'm confused!

I don't know how it happened. but there is a minimal chance to happen this because the ligase buffer contains ATP and there might happen some spontaneous reaction to phosphorylate the insert. Its just chance. I think its not possible to get desired colony every time you perform the reaction without phosphorylating your insert. I tried several times and got it...

#8 Papaver

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Posted 12 July 2012 - 04:21 AM

I would say, as long as you only need one (right) clone, chances are good. It's not the best way if your cloning is complicated.
For me it also worked well but I only had to clone a resistence gene into a plasmid...




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