Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

Problem with site directed mutagenesis - (Sep/24/2009 )

I had some problem about Site directed mutagenesis(SDM). Anybody can help me,please.


I used quik-change Site directed mutagenesis(SDM) kit to did mutant(single amino acid change).
But Tm of my primers not up to 78oC. I can see a little band on my gel when I ran agarose gel
What way can I increase Tm of my primers and increase my PCR products.


This is my primers:

1.F-primers : B_N164A_F 5- ATC ACT CCC TGG GCT TAT CCT CTG CTG -3
R-primers : B_N164A_R 5- CAG CAG AGG ATA AGC CCA GGG AGT GAT -3
Tm = 71.87 oC

2.F-primers : O_N162A_F 5- ATC ACA CCT TGG GCC TAT CCT CTC CTG -3
R-primers :o_N162A_R 5- CAG GAG AGG ATA GGC CCA AGG TGT GAT -3
Tm = 71.87 oC


This is my conditions:


Stock concentration Final concentration Volume(ul)
DNA template (50ng/ml) 100 ng 2
F-primer (10uM) 125 ng 1.5
R-primer (10uM) 125 ng 1.5
dNTPs(2.5mM) 0.2 mM 4
10X buffer 1X 5
Sterile dd.H2O 36
Total volume 50

Then add pfu 2.5U and then did the PCR.

When PCR finished I added DpnI and incubate follow quik-change protocol.

After incubated I ran agarose gel.


This is my Themo cycling :

segment temperature time cycle
1 94oC 30 sec 1
2 94oC 30 sec 16
55oC 1 min
68oC 14 min
3 72oC 12 min 1

p.s. segment 2, I used 16 cycle.



Anybody can help me,please. ;)

Caren

-caren-

Ist try to optimimize your PCR parameters.......or consult the kit manufacturers, they will provide you assistance and directions on what to do............ The only way to increase your primers Tm is to redesign them by adding some more bases......Try to do it with the software that is on the website of the kit.....they will give u primers that are in accordance with requirements.....but some times it has problems of self complementarity and hair pin formation..........
if u increase the length of your primer to 38-42 depending on the GC content u will attain the required Tm.......what about your primers?are they PAGE/HPLC purified?
have you transformed?
dnt trust on the gel in this case as the kit manual (if its from stratagene) say u will observe it on gel or not but proceed towards transformation........after transformation u can decide about your pcr.....

-Adnan-

Thank you for your suggestion, Adnan. :P

I transformed my PCR product already. But I didn't get any colony.
May I decrease annealing temp?
May my PCR product will increase?

-caren-

caren on Sep 25 2009, 05:39 AM said:

Thank you for your suggestion, Adnan. :)

I transformed my PCR product already. But I didn't get any colony.
May I decrease annealing temp?
May my PCR product will increase?

Dear caren,
I have recently mutegenized my construct with this kit.I think something is wrong with the competent cells which have been provided with the kit.I tried our own DH5a competent cells and I got plenty of colonies.Also increase the amount of Dpn1 treated PCR product from 1ul(according to protocol) to 10 ul.if it did n't work then you can conclude that something is wrong with PCR.

-banou-

Dear caren,
I have recently mutegenized my construct with this kit.I think something is wrong with the competent cells which have been provided with the kit.I tried our own DH5a competent cells and I got plenty of colonies.Also increase the amount of Dpn1 treated PCR product from 1ul(according to protocol) to 10 ul.if it did n't work then you can conclude that something is wrong with PCR.




What kit that you use? STRATAGENE?
I think my problem may be from transformation and PCR condition.
I see a little band PCR product after add DpnI.
May be my mutant not re-ligate?
I'll try to do it again.

Thanks a lot!! banou. :)

-caren-

Yes I have used the kit from stratagene.In my idea it's better to chek your transformation efficiency with another vector from your lab and also trying anothe type of competent cells.when you see a band so the PCR have worked and about the religation I think it's not the case.

-banou-

I use the kit from stratagene and I have no problem. Try this tips:
I add at least 100ng of plasmid.
I saw that you use 2.5ul of Pfu. I think with 1ul is enough.
Did you use enough time in the last step of your pcr? sometimes that is the problem. Remember that you have to use more time if your plasmid is bigger otherwise you wont get a complete amplification.
1ul of DpnI during 1h is enough.
(At this point you dont have to see a product in a gel)
Try to use 2-5 ul of your sample to do the transformation in 50ul of competent cells

-laurequillo-

Yes u can try but by decreasing annealing temperature your product wnt be that specific..........as it is on high annealing temprature.
U havnt told about the kit? what specific type is this kit? i also had problems with kit from stratagene.....that was quickchange lightning multisite directed mutagenesis kit..........what are u using? regular kit or the one i mentioned? give some more information and then can i tell you......

-Adnan-