Site-directed Mutagenesis - (Jun/28/2006 )

Hi,

I would like to know whether somebody here made some bad experiences with the Quick-Change Site Directed Mutagenesis Kit from Stratagene, too.
I have performed now many reactions with different amounts of template DNA as it is recommended in the manual but I only get some clones after transformation and the effiency of mutagenesis is also very low. About 20% of all clones carry the mutation. It should be about 90% as it is for the control reaction which works really perfect.
So, where is the problem? Could it be that vector itself influences the reaction? The control vector is pWhitescript but I used Gateway compatible vectors for my reaction.
It would be nice if somebody here has an idea to this problem, because this Kit from Stratagene is really expensive.

Thanks in advance.

Klaas

Hi Klaas,

we don'øt buy the kit but the polymerase and the DpnI enzyme and it's much cheaper.
This are the conditions I use:
Reagents amount
10x HF-Buffer 5 µl
25mMdNTP 1µl
for-Primer (125 ng/µl) 1 µl (125 ng)
rev-Primer (125 ng/µl) 1 µl (125 ng)
PfuUltra-Polymerase 1 µl (2,5U)
dH20 40,5 µl
DNA (100 ng/ µl) 0,5 µl (50 ng)
final volume DNA included 50 µl



Temperature Time
1 Denaturation 95°C 30s


2 (18cycles) Denaturation 95°C 30s
Annealing 55°C 1 min
Elongation 68°C 14min (2min/Kb plasmid)

3 Cooling 4°C

Hi lusi,

I'm with DNA factory, I commonly do mutagenesis and don't use a kit only the DpnI and a good long range proofreader (my preference at the moment is Accuprime Pfx). The efficiency of mutation is dependant on a number of things including the size of plasmid, the efficiency of your DpnI treatment and the design of your primers and of course the actual sequence. For instance one of the mutations I'm doing at the moment is proving to be dificult due to a large A/T rich region and I am having to go back to a Splice Overlap Mutation. However, I would always try the quickchange method first for the speed and ease.

The principle is quite simple you design primers containing the mutation and areas of homology 5' and 3' of the desired mutation. The PCR reaction amplifies the whole vector. You then digest the parental vector using DpnI (this digests methylated DNA, PCR product is not modified and therefore is not digested). Transform and resultant colonies should have your mutation.

Hope this helps

Cheers,

Scott

For your large AT rich region, try the PCR with a low extension (not annealing) temperature. 62-66C, for example.

Thanks for the tip Phage434, will give it a go.

Cheers

Thanks, Scott!

It helps more in my understanding of mutagenesis, but hope that I don have to do tat, cos actually I am currently doing a small project in school (I am a student) and cos time & fund is limited, I hope I can get the exact insert that I have without having to do site directed mutagenesis.

Primer extension can only happen at 3' ends bound to a 5' overhang. Since the mutageneic primers are fully complementary, although they will bind to each other, there will be no extension. Since they are in vast excess, some will bind to the target, where extension will take place.