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

invitro mutagenesis using stragene kit - (Mar/29/2011 )

Hi

Iam having problem in invitro mutagenesis. I am using kit from stragene. I did the cycle according to the maufacturers instruction. But nothing is working, even after repetitions. I designed the primers in their website. I didnt note the TM of the primers while designing. The primer of the Tm is around 68 deg according to the data sheet. The conditions used are

5 μl of 10 reaction buffer
X μl (10 ng) of dsDNA template
X μl (125 ng) of oligonucleotide primer #1
X μl (125 ng) of oligonucleotide primer #2
1 μl of dNTP mix
3 μl of QuikSolution
ddH2O to a final volume of 50 μl

1 l of PfuUltra HF DNA polymerase (2.5 U/l)




95C 1 minute
95C 50 seconds
60C 50 seconds

68C 1 minute/kb of plasmid length, 18 cycles
68C 7 minutes


Will it work if I set the TM for the primer according to the data sheet, or should I go with the TM given by the manufacturer.

Any help will be greatly appreciated.


Regards,

jaya

-jaya2020-

I know they have several different mutagenesis kits from Stratagene/Agilent, and I use the Quikchange II Mutagenesis Kit, so I don't know how helpful this will be to you. But I believe they give you an equation in the protocol for calculating the Tm of the primer. I would use that equation. It might take a little time to calculate it, but I've designed primers making sure that the Tm, calculated using that equation, is at or above 78 deg. C as the manufacturer's designate. I've always gotten good results with the mutagenesis making sure that my Tm is in that range.

So, I might suggest that you calculate the Tm with that equation, and see if it falls above 78 deg C.

I notice your PCR settings are different from the one that I've used. I don't know if it's because you have a different kit, but check out what the manufacturer's protocol says again, and make sure you're not deviating too much from it.

-kmh-

kmh on Wed Mar 30 01:09:54 2011 said:


I know they have several different mutagenesis kits from Stratagene/Agilent, and I use the Quikchange II Mutagenesis Kit, so I don't know how helpful this will be to you. But I believe they give you an equation in the protocol for calculating the Tm of the primer. I would use that equation. It might take a little time to calculate it, but I've designed primers making sure that the Tm, calculated using that equation, is at or above 78 deg. C as the manufacturer's designate. I've always gotten good results with the mutagenesis making sure that my Tm is in that range.

So, I might suggest that you calculate the Tm with that equation, and see if it falls above 78 deg C.

I notice your PCR settings are different from the one that I've used. I don't know if it's because you have a different kit, but check out what the manufacturer's protocol says again, and make sure you're not deviating too much from it.



Thank you for the reply. I have the same kit as u mentioned in my lab. I will try using that. The Tm primer that I have now was designed in the software that they had recomended. I notice the Tm of the primer is around 68 deg. Since I used the online program, I did not give importance to the Tm as the protocol says. Is there a program to design the primer with the required mutation, or do I have to do it manually. Can u also please give me the protocol that u had used.

Thanks,

jaya

-jaya2020-

I designed my primers manually going by what the manufacturer's manual/protocol states. You might want to try it that way.

This is the manual. Page 6 is what you want to look at. It tells you everything you need to know about the primer design including that equation I was talking about.
http://www.genomics.agilent.com/files/Manual/200523.pdf

It might take extra time designing it manually, and you'll have to be careful to make sure your sequences are correct, but it should give you a good primer for the mutagenesis.

-kmh-

kmh on Wed Mar 30 16:08:52 2011 said:


I designed my primers manually going by what the manufacturer's manual/protocol states. You might want to try it that way.

This is the manual. Page 6 is what you want to look at. It tells you everything you need to know about the primer design including that equation I was talking about.
http://www.genomics.agilent.com/files/Manual/200523.pdf

It might take extra time designing it manually, and you'll have to be careful to make sure your sequences are correct, but it should give you a good primer for the mutagenesis.



Thank you for the reply. I will do as per the manufacturers instructions.

regards,

jaya

-jaya2020-