Low mutation efficiency - (Jan/31/2007 )
Hi everyone!
I am using the quick change Kit (stratagene) and my problem is that I obtain colonies but all the colonies I have sequenced do not carry the 3 mutations I want to insert. ¿What could be the problem? I test DpnI and it works well, my primers length is 37bp
Please give me advices
hey please I am very stressed I made my PCR reaction as follows:
DNa template 50 ng
Primer1 150 ng
Primer2 150 ng
1 ul 25 mM dntps
1 ul Pfu
H20 to 50 ul
95°C 2 min
95°C 40 sec
55°C 1 min
72°C 6 min ( my plasmid is of 5.5 Kb)
72°C 10 min
then I added 10 U of Dpn I and incubated for 1 hour at 37°C, after that I transformed 2 ul of the reation and I obtained 86 colonies, I screened 25 and any of the colonies carried the desired mutation
¿What can it be wrong?
Please I thank all suggestions
check ur primer design
i use kit from takara, they have a mgcl2 sol include in the kit
did you use buffer in your pcr reaction?
how many cycles?
did you confirm the result of the pcr before dpn treatment?
[quote name='mdfenko' date='Feb 2 2007, 08:50 AM' post='86563']
did you use buffer in your pcr reaction?
how many cycles?
did you confirm the result of the pcr before dpn treatment?
Yes I did use 5 ul of 10 X PCR reaction
and I did confirm the PCR reaction on an 1% agarose gel I saw the band of the right molecular wight, so I procced with dpn I digestion and transformation but I did not get colonies with the mutation
What coul it be happening?
Thanks
I made 16 cycles of the PCR reaction
did you confirm the result of the dpn treatment prior to transformation?
Yes I checked it and I did not observed any band, so I tought that all my template was digested and I have just few copies made in the PCR,so I transformed and I did obtain colonies but without the mutation
