6 replies to this topic

[bitpas]

I want to prepare a blunt sequence using T4-Polymerase:

The Sequence is  xxxCTGCA            (PstI, after digest)
...........................xxxG

It is very important for my experiment that I keep the C, eg:  xxxC          
to stay in frame!......................................................................xxxG

Does anyone have a routinely working protocol for this reaction?
Did ever someone of you do something like that?

or any other possibilities to get rid of the "sticky overhang"?

Every response is very welcome!!  

Edited by bitpas, 23 August 2004 - 01:47 AM.

[blasko]

Why don't you try Klenow + dntp?
37 C, 15 min.

[bitpas]

Thanks for your replay!!
sorry, the sequence was wrong.. now its right.

Klenow: If i just fill the "sticky overhang" i would loose the frame for  the cloning afterwards.  Anyway Klenow (or other "fillers") can't be used since the overhang is 3'.

[lyrezxl]

in the case like this, i always just use Mung Bean Nuclease.
for

Quote

It is very important for my experiment that I keep the C, eg: xxxC
to stay in frame!......................................................................xxxG

i'm not sure it:o
but in our cases, after MBN digestion, the sequence is always just what we need

hope it helps!

lyre

[bitpas]

@lyrezxl
yes, MBN seems to be more suitable for this reaction! Thanks!

Do you have a protocol which you could share?

Greetings!

[lyrezxl]

Hi,
the MBN i used are from Takara and Promega. both of them work very well.
and i just do it as they recommended.
good luck!
lyre

[arwen76]

I'm trying the same with KpnI. To remove the overhang you need to use T4 DNA Polymerase...And also dNTPS