Can pfu Polymerase make a blunt end? - (May/21/2006 )

1.Could pfu(MBI) be chosen to fill in 5` overhang and cut 3`overhang?

2.If pfu can cut the cohensive end,what is the optimal temp.?

3.If my vector have a blunt end and a sticky end, does dephosphorization still needed before ligation?

Need ur help. Many thinks!

---

1. pfu is a proof-reading polymerase and will hence give you blunt ends
2. optimal temp for amplification with pfu is the same as taq, 72 c
3. a blunt end will in principle, not ligate to the sticky end

all the best

-viv

---

[quote name='viv' date='May 22 2006, 01:33 AM' post='52662']
1. pfu is a proof-reading polymerase and will hence give you blunt ends
2. optimal temp for amplification with pfu is the same as taq, 72 c
3. a blunt end will in principle, not ligate to the sticky end

all the best

Thank you so much!

---

----------------------------------------------------------------------------------------------------------------

I have to clone 1.4Kb promoter of gene. I used home made taq polymerase (no proof reading) its working very well and i applied same condition with pfu (stratagene) but its nor working at all.

I dont know at this point what shall i do, for pfu i used 95-5mins and 94-45sec,58-45sec, 72-2.5mins for 30 cycles, then 72-10mins as a final extension

if any one can guide me?

Imteyaz

---