cloning doubt

[cloningproblem]

Hi,

i hav a Pcr product for site EcoRI and Sma I but it has got by using taq polymerase (EcoRI-cohisive site) and Pfu polymerase (Sma I-blunt site) then digest Pcr product with EcoRI and gel purified the product was ligated with vector sequencially digested with Sma I and EcoRI gel purified with good DNA concentration, however after ligation could not get any transformed colonies,

i hav this doubt that, while using Pfu tag would it removes EcoRI site of PCR product so that the ligation fails are any other incompactibility may there,

how can i do clone the EcoRI / Sma I pcr product in vector digested with Sma I and EcoRI ?

[mmorgan]

Originally Posted by cloningproblem

Hi,

i hav a Pcr product for site EcoRI and Sma I but it has got by using taq polymerase (EcoRI-cohisive site) and Pfu polymerase (Sma I-blunt site) then digest Pcr product with EcoRI and gel purified the product was ligated with vector sequencially digested with Sma I and EcoRI gel purified with good DNA concentration, however after ligation could not get any transformed colonies,

i hav this doubt that, while using Pfu tag would it removes EcoRI site of PCR product so that the ligation fails are any other incompactibility may there,

how can i do clone the EcoRI / Sma I pcr product in vector digested with Sma I and EcoRI ?

Are you using both Taq and Pfu together?

I would just use Pfu (or Pfx, anything that will create a blunt end) and then cut with EcoRI. Taq will add 3' overhangs to your PCR product.

Also with your strategy if you de-phosphorylate your cloning vector the cloning will not work.

PCR primers don't usually have 5' phosphates (unless you pay extra for them) so your SmaI-blunt end won't ligate if you have de-phosphorylated your plasmid.

If you have EcoRI and SmaI sites in your PCR primers, you could cut your product with EcoRI-SmaI and clone it. Better still you could use XmaI instead of SmaI (they cut the same site except XmaI gives you a cohesive end).

[cloningproblem]

Dear mmorgan

i could not understand your post reply, can you please explain things detail, and i explain my doubt one second here that i have got my insert by using taq polymerase and Pfu polymerase together in PCR reaction because my forward primer was ECoRI (cohisive type) and reverse primer was Sma I (blunt type), if i use taq pol alone i can't have blunt end as it add 'A', if i use pfu taq alone i can't get ECORI site at 5' end hence i used two types of polymerase (0.5µl+0.5µl),

how should i do PCR using ECoRI site forward primer and Sma I site reverse primer, what polymerase enzyme should i use and i cut my vector with 5' ECoRI and Sma I 3' enzymes sequencially and gel purified the vector without dephosphorylated...