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Blunt ligation with PCR: is kinase needed?


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11 replies to this topic

#1 lna

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Posted 24 April 2009 - 12:44 AM

Hi,
sorry for the question, but I'm somehow confused here.. I have the vector, cut by NotI. And a PCR product, made by Taq (primers not phosphorylated). What I need - to stick them together. So, if I get it right, the following steps should be done:
make the vector's ends blunt by T4 pol, make the PCR product blunt with the same T4, phosphorylate the PCR by kinase and perform the ligation... Seems to be rather unefficient protocol, but I want to try before ordering the new primers with NotI etc.
So, the question is - do I really need to phosphorylate the PCR product if I do not treat the vector with CIAP? Is it so necessary to treat the PCR product with T4 pol - may be there are soome blunt-ended products present evenwith Taq, which could work for the ligation?
Am I reasonable here anyway?
Thank you in advance for any hints!

#2 little mouse

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Posted 24 April 2009 - 12:55 AM

About the PCR product, I don't know, but about not dephosphorylate the vector : I know you will make a mistake. If you don't dephosphorylate the vector you will have plenty of empty vector. Believe me, treat the vector with CIAP. I always dephosphorylate the vector, because I already have very bad luck with insertion in the wrong way. Then , I don't want to have plenty of empty vectors too !

#3 lna

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Posted 24 April 2009 - 01:10 AM

About the PCR product, I don't know, but about not dephosphorylate the vector : I know you will make a mistake. If you don't dephosphorylate the vector you will have plenty of empty vector. Believe me, treat the vector with CIAP. I always dephosphorylate the vector, because I already have very bad luck with insertion in the wrong way. Then , I don't want to have plenty of empty vectors too !


so, if I CIAP the vector, then I defifnitely need to treat the PCR product with the kinase - am I right?

#4 Bomber

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Posted 24 April 2009 - 03:09 AM

Yes, yoou really should phosphorylate yoour pcr product.
Not cipping the vector will only result in almost 100% religation (see little mouse's post) since the two ends are in close proximity in the ligation reaction.
The safest option is to cip the plasmid and to phosphorylate the pcr product.

Good luck

#5 T C

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Posted 24 April 2009 - 03:21 AM

Hey,

What are you doing this for?

If the site is not an issue but the vector is then just religate the vector and clone this blunt fragment in a blunt site like Eco RV, Stu I or Sna BI.

As discussed above, you need to CIP the vector and phosphorylate the insert unless yr primers are phosphorylated.

Best,
TC

Hi,
sorry for the question, but I'm somehow confused here.. I have the vector, cut by NotI. And a PCR product, made by Taq (primers not phosphorylated). What I need - to stick them together. So, if I get it right, the following steps should be done:
make the vector's ends blunt by T4 pol, make the PCR product blunt with the same T4, phosphorylate the PCR by kinase and perform the ligation... Seems to be rather unefficient protocol, but I want to try before ordering the new primers with NotI etc.
So, the question is - do I really need to phosphorylate the PCR product if I do not treat the vector with CIAP? Is it so necessary to treat the PCR product with T4 pol - may be there are soome blunt-ended products present evenwith Taq, which could work for the ligation?
Am I reasonable here anyway?
Thank you in advance for any hints!



#6 lna

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Posted 24 April 2009 - 03:40 AM

Hey,

What are you doing this for?

If the site is not an issue but the vector is then just religate the vector and clone this blunt fragment in a blunt site like Eco RV, Stu I or Sna BI.

As discussed above, you need to CIP the vector and phosphorylate the insert unless yr primers are phosphorylated.

Best,
TC

Hi,
sorry for the question, but I'm somehow confused here.. I have the vector, cut by NotI. And a PCR product, made by Taq (primers not phosphorylated). What I need - to stick them together. So, if I get it right, the following steps should be done:
make the vector's ends blunt by T4 pol, make the PCR product blunt with the same T4, phosphorylate the PCR by kinase and perform the ligation... Seems to be rather unefficient protocol, but I want to try before ordering the new primers with NotI etc.
So, the question is - do I really need to phosphorylate the PCR product if I do not treat the vector with CIAP? Is it so necessary to treat the PCR product with T4 pol - may be there are soome blunt-ended products present evenwith Taq, which could work for the ligation?
Am I reasonable here anyway?
Thank you in advance for any hints!


in fact, the site is important - NotI cuts my vector two times, so I can replace the insert and incorporate my gene of interest between the promoter and polyadenlation signal. Thank you for the answers! so, gone searching for kinase..

#7 mastermi

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Posted 24 April 2009 - 08:04 AM

Why don't you use primers with NotI sites, cut the PCR product and make a sticky end ligation?

#8 lna

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Posted 26 April 2009 - 11:58 PM

Why don't you use primers with NotI sites, cut the PCR product and make a sticky end ligation?

I just did not wanted to order the additional primers. moreover, I've already tried to do the ligation of this sequence with another restriction sites-containing primers (into another vector) and it was not working (do no know why).

#9 perneseblue

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Posted 27 April 2009 - 03:00 AM

Why don't you use primers with NotI sites, cut the PCR product and make a sticky end ligation?

I just did not wanted to order the additional primers. moreover, I've already tried to do the ligation of this sequence with another restriction sites-containing primers (into another vector) and it was not working (do no know why).


Was the NotI site skirted with enough bp? NotI site requires a minimum of 6bp before it will be cut efficiently
May your PCR products be long, your protocols short and your boss on holiday

#10 lna

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Posted 27 April 2009 - 06:38 AM

Why don't you use primers with NotI sites, cut the PCR product and make a sticky end ligation?

I just did not wanted to order the additional primers. moreover, I've already tried to do the ligation of this sequence with another restriction sites-containing primers (into another vector) and it was not working (do no know why).


Was the NotI site skirted with enough bp? NotI site requires a minimum of 6bp before it will be cut efficiently


it was not the Not site that I used before (because I was trying to insert into another vector). I used BamHI and XbaI and I left 4 additional nt on the ends - to my knowledge this should be enough for these enzymes..
Thank you for your interest. Actually, I was trying to verify here, whether the kinase reaction is needed to ligate the PCR product into the cut vector.

#11 hanming86

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Posted 29 April 2009 - 07:24 AM

Why don't you use primers with NotI sites, cut the PCR product and make a sticky end ligation?

I just did not wanted to order the additional primers. moreover, I've already tried to do the ligation of this sequence with another restriction sites-containing primers (into another vector) and it was not working (do no know why).


Was the NotI site skirted with enough bp? NotI site requires a minimum of 6bp before it will be cut efficiently


it was not the Not site that I used before (because I was trying to insert into another vector). I used BamHI and XbaI and I left 4 additional nt on the ends - to my knowledge this should be enough for these enzymes..
Thank you for your interest. Actually, I was trying to verify here, whether the kinase reaction is needed to ligate the PCR product into the cut vector.


I have not used kinase throughout my time doing cloning. u only need one phosphorylated end on both side. and then the E.coli have mechanism to gap repair the unligated portion of it.



P-------OH
OH-----P

after ligation

----OH-P-------OH OH-------
----OH OH------P-OH--------

after E.coli

----OH-P-------OH-P-------
----P-OH------P-OH--------
Lab + Coffee + Music = Bliss

#12 lna

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Posted 05 May 2009 - 01:53 AM

Why don't you use primers with NotI sites, cut the PCR product and make a sticky end ligation?

I just did not wanted to order the additional primers. moreover, I've already tried to do the ligation of this sequence with another restriction sites-containing primers (into another vector) and it was not working (do no know why).


Was the NotI site skirted with enough bp? NotI site requires a minimum of 6bp before it will be cut efficiently


it was not the Not site that I used before (because I was trying to insert into another vector). I used BamHI and XbaI and I left 4 additional nt on the ends - to my knowledge this should be enough for these enzymes..
Thank you for your interest. Actually, I was trying to verify here, whether the kinase reaction is needed to ligate the PCR product into the cut vector.


I have not used kinase throughout my time doing cloning. u only need one phosphorylated end on both side. and then the E.coli have mechanism to gap repair the unligated portion of it.



P-------OH
OH-----P

after ligation

----OH-P-------OH OH-------
----OH OH------P-OH--------

after E.coli

----OH-P-------OH-P-------
----P-OH------P-OH--------


thank you! I guess, this answers my question directly! (:




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