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Cloning a 60 bp pcr product and Mixing Pfx and Taq polymerases - (Jun/26/2007 )

Hi all,

I have two questions

1]I have a cDNA template which can be amplified only with pfx and i want to clone it by TOPO-TA cloning. Can i add some Taq to the pfx while i do the pcr to add dA over hangs to the final product? If yes, howmuch should i add? also should i add Taq specific buffer to the pcr mix too? if yes, what is the proportion?


2] I get a faint 60 bp band upon pcr. I need to clone it. What pecentage of the total pcr product (say 50 ul) should i use for ligation. Looks like there is little pcr product and TOPO-TA manual says to use 0.5ul to 4 ul of the pcr product for ligation. But i feel that 4 ul is not enough considering that i get very little pcr product.


thanks in advance for your advices.

-polsum-

QUOTE (polsum @ Jun 26 2007, 09:08 PM)
Hi all,

I have two questions

1]I have a cDNA template which can be amplified only with pfx and i want to clone it by TOPO-TA cloning. Can i add some Taq to the pfx while i do the pcr to add dA over hangs to the final product? If yes, howmuch should i add? also should i add Taq specific buffer to the pcr mix too? if yes, what is the proportion?


The way I do it is to column purify the PCR fragment from the proof reading polymerase. Then I set up a reaction with Taq buffer, only dATP and Taq. I then incubate the lot at 72 Celsius for 30mins. If the proof reading polymerase is around it will do its best to remove that A overhang that Taq adds on.



QUOTE (polsum @ Jun 26 2007, 09:08 PM)
2] I get a faint 60 bp band upon pcr. I need to clone it. What pecentage of the total pcr product (say 50 ul) should i use for ligation. Looks like there is little pcr product and TOPO-TA manual says to use 0.5ul to 4 ul of the pcr product for ligation. But i feel that 4 ul is not enough considering that i get very little pcr product.


The TOPO cloning is very efficient. But Taq is not. At the very best Taq only adds A tails to 70% of the PCR product. I would concentrate the PCR product and throw in about 100ng (yeah I know, I am being excessive.) of the PCR product.

-perneseblue-

I do the same as Pernesse, column purify PCR product and then proceed with adding the A-overhang with taq.

How much colonies do you need? You might be lucky and try with 4 ┬Ál of PCR product if you just need one.

-vairus-

QUOTE (perneseblue @ Jun 26 2007, 02:39 PM)
QUOTE (polsum @ Jun 26 2007, 09:08 PM)
Hi all,

I have two questions

1]I have a cDNA template which can be amplified only with pfx and i want to clone it by TOPO-TA cloning. Can i add some Taq to the pfx while i do the pcr to add dA over hangs to the final product? If yes, howmuch should i add? also should i add Taq specific buffer to the pcr mix too? if yes, what is the proportion?


The way I do it is to column purify the PCR fragment from the proof reading polymerase. Then I set up a reaction with Taq buffer, only dATP and Taq. I then incubate the lot at 72 Celsius for 30mins. If the proof reading polymerase is around it will do its best to remove that A overhang that Taq adds on.



QUOTE (polsum @ Jun 26 2007, 09:08 PM)
2] I get a faint 60 bp band upon pcr. I need to clone it. What pecentage of the total pcr product (say 50 ul) should i use for ligation. Looks like there is little pcr product and TOPO-TA manual says to use 0.5ul to 4 ul of the pcr product for ligation. But i feel that 4 ul is not enough considering that i get very little pcr product.


The TOPO cloning is very efficient. But Taq is not. At the very best Taq only adds A tails to 70% of the PCR product. I would concentrate the PCR product and throw in about 100ng (yeah I know, I am being excessive.) of the PCR product.



What columns should i use to purify/concentrate such short pcr product. Qiagen pcr purification kit has a range from 100 - 20kb. Are there any other commercial columns which i can use here?

-polsum-

Ah 60bp... somehow I though it was a typo and meant 600bp. blink.gif
Well, you can buy 2, 60bp oligomere and annealing them togther yourself or request that the oligomere be annealed for you by the company. It saves you the hassle of doing your own PAGE purification

Er.. I hope this 60bp fragment is not related to the cDNA template story.

-perneseblue-