Site-directed Mutagenesis - (Jun/28/2006 )
ThX! phage. I C.
This are the conditions I use:
10x HF-Buffer 5 µl
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
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
Would this condition works with large plasmids as well? I'm working with 14 kb and got no colony from Stratagene, QuikChange II XL SDM kit. Any tips for the big plasmids? Do you usually see the band after PCR? Do you have to concentrate the DNA after DpnI digestion? If not how much do you use for transformation?
I'm using this kit right now and it works with me almost witth 100% efficiency. All the colonies I sequenced carried the mutation desired. I usually get around 100 colonies from 250 ul on the plate.
I use 20 ng of plasmid and it works fine.
what about your primers? how is the Tm? and length?
I usually use 34mer primers HPLC purified and I follow the instructions exactly.
I used the kit with more than 12 plasmid and they worked perfectly.
need anything let me know
I would recomend reading the following article:
the change in primerdesing is only small, but has great effect. Up to now I had success with this methode in 100% of experiments.
If quikchange is not working for you, I would try SLIM PCR.
Here is a good article:
The only alteration I make is that I perform the two overlapping regions in separate tubes to maximize proper heterodimerization formation on the second step.
Generally I use quikchange first, but SLIM PCR is probably more efficient (although it requires more primers).
I am just about to design my primers for my 1st mutation with the Quick Change kit, so it's great to read you.
I am planning to use 31 nt inserts (Tm sounds alright). Do you think that it's necessary to use purified (HPLC or PAGE) primers?
According to Stratagene you have to expect a 25% decrease in efficiency when using non-purified primers, so I used standard desalted PCR primers (they were much cheaper) and I got no problems, I got plenty of colonies and most of them carrying the desired mutation.
I have been trying to do SDM for last one week.Problem is , I am not getting colonies after transformation except for one mutated plasmid. I am confused. what can be the possible cause?
no mutation( I saw bands after gel run)
bad transformation ( I used calcl2 treated competent cells)
I used pfu ultra( some say, turbo would be better!!)
I am doing loop mutations
I used almost 20 unit DpNI for digestion.
Can you please help me????
I've used PfuUltra for SDM and it worked like a charm, so I doubt that would be the problem.
What do you mean with "one mutated plasmid"? Is it a plasmid with your desired mutation, or with an extra mutation elsewhere or only with a mutation elsewhere?
Something that has worked for me doing SDM on quite large plasmid was to do the PCR and the digestion (more DpnI and 2 hours longer than stratagene's protocol, just to make sure), then purify it (column based, elution in 50 µl of water) and I vacuümed up my reaction to about 20 µl, then ligated it (about 10 µl of the eluate in a 20 µl reaction) transformed 5 µl of it, got a lot of colony's... (transforming Dh5alpha, no special bacteria whatsoever)
What worked for me was the PCR with 68°C 2min/kb plasmid, purify and take up in amount of water needed for standard 20µl digest, then stop reaction and purify and then transform 2-6 µl (out of 50µl). With this I got nice colonies on my plates. All other attempts just failed.