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Jason_LuYZ

Member Since 16 May 2012
Offline Last Active Apr 16 2013 08:45 PM

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kartiga natarajan
21 Feb 2013 - 08:19
AmitS
24 Sep 2012 - 18:09
anf_zahra
31 Jul 2012 - 00:15

#134477 Site-directed mutagenesis

Posted Alisa on 13 May 2012 - 07:49 PM

Hi, I've done mutagenesis for 3 month, but until now all of my colonies are wild type. My vector is pDEST26 (7.3kb) and target gene is 3kb ,so total gene is about 12kb. I've try lots of conditions:

1. Used

Stragene QuikChange II site directed mutagenesis Kit

10x reation buf.      5ul
DNA template      50ng
f-primer                  1ul
r-primer                  1ul
dntp                        1ul
ddH2O                  36ul
pfu                          1ul
TOTAL                 50ul

95

℃-->1 min
95℃-->30sec
55℃-->1min
68℃-->12min (30cycles)

68℃-->5min

DpnI digest for 1hr, add 4ul DpnI digested product to 50 ul competent cell(DH5a)

2ml SOC incubate 6hr

incubate on plate(amp+)

@this condition I have lots of colonies but they are not mutant!

2. Used

Stragene QuikChange II site directed mutagenesis Kit

I asked Stragene. They suggest that I can raise anneal tem. to Tm-3

℃.
So I change my tem to 62℃, again I can't get any mutatant!

3. Use pfu from fermantas and change primer (design from

Stragene:

QuikChange Primer Design)

I can't get any mutatant again!

I am no idea now!!

One of my friends said that I should use

Stragene's pfu (for large vectors), it's that true? But it's so expensive!!

Anyone who has better ideas please tell me!
Thank you!