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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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Site directed mutagenesis with PCR
Posted by: nit (IP Hidden, New member, 4)
Date: June 19, 2005 03:32AM
Hi
I want to introduce three enzyme sites and an RBS site in my plasmid. So i decided to carry out site directed mutagenesis wherein with one pcr I would be able to introduce all the sites. I designed the forward and reverse preimers correspondingly. But now i am not too sure about the condition i should maintain during my pcr. I tried with the standard protocol given in the stragene kit (which i used) but i dont think it worked. The melting point of my primer is 87.5' C. Also I am not able to get any colonies on transformation. It would be great if some one could point out the condition and the error. I need to be done with my work in a few weeks time. Any thought will be greatly appreciated. Thanks...
Re: Site directed mutagenesis with PCR
Posted by: John H (IP Hidden, Unregistered user, )
Date: June 20, 2005 07:04PM
Did you get any colonies with the control reaction? Ensure the control is working and then check if the primer design is correct.
Re: Site directed mutagenesis with PCR
Posted by: nit (IP Hidden, New member, 4)
Date: June 21, 2005 01:23AM
Hi John
Thanks. But what exactly do you mean by control? I did keep a control of only DNA and water mixture for PCR and was unable to get any colonies. So I prepared fresh plasmid DNA by miniprep kit. Can you please suggest me the conditions to be maintained for this site directed mutagenesis as my primer melting point are 87.5' C.
Re: Site directed mutagenesis with PCR
Posted by: John H (IP Hidden, Unregistered user, )
Date: June 21, 2005 08:29AM
I mean the positive control provided by the kit. It may help you to determine if the problem araise from the pcr reaction, transformation etc other than primer design itself. The Tm you mentioned seems ok since the kit requires >=78c. Any thoughts?
Re: Site directed mutagenesis with PCR
Posted by: Dan (IP Hidden, Unregistered user, )
Date: June 21, 2005 05:13PM
I can only say I had several troubles to mutagenize big plasmids and this gets much better increasing the elongation time (25 min for 10 KB) and slightly decreasing annealing temperature (58-59 deg). Are you planning to introduce three sites all at once??? Do you have primers long enough (at least 20 bp) on each side of the mismatch??
Precipitation of the PCR reaction and transformation of more DNA helps as well and I usually get transformants even if the PCR product is not detectable on gel. Are your primers PAGE purified? If you do not get the desired mutations, why do not try to introduce your mutation in sequent steps? Good luck
Re: Site directed mutagenesis with PCR
Posted by: nit (IP Hidden, New member, 4)
Date: June 22, 2005 12:51AM
My plasmid is only 3.7kb. Ya i am planning to introduce all the three sites at once. As i told earlier my primer is about 57 bases long and has a melting point of 87.5' C. The conditions I have maintained for the PCR are same as that given in the Stratagene book. Conditions as follows:
1) 95 degree C for 30 sec 2) 95 degree C for 30 sec 3) 55 degree C for 1 min 4) 68 degree C for 4 min 5) 4 degree for infinity time. The steps 2-4 are repeated 18 times ( as given in the book for addition). Please let me know if I have to make any changes in the protocol Thanks.
Re: Site directed mutagenesis with PCR
Posted by: Dan (IP Hidden, Unregistered user, )
Date: June 22, 2005 02:01PM
Dear Nit,
I realized that extension time is very important! you should increase the extension time, I would do 10 minutes elongation time for your plasmid. And stick to the conditions reported on the manual 1) 95 deg for 1 min 2)95 deg for 50 sec 3)58-60 deg for 50 sec 4)68 deg for 10 min 5)68 deg for 15 min 6) 4 deg infinite repeat 2-4 for 18 times. Try to increase the amount of template up to 50 ng and keep primer concentrations as indicated on the manual (125 ng each) I hope this can help. It worked for me and I wish you good luck...
Re: Site directed mutagenesis with PCR
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: June 24, 2005 08:02AM
4 minutes may not be long enough for the extension step. try increasing it to 10-15 minutes.
Re: Site directed mutagenesis with PCR
Posted by: nit (IP Hidden, New member, 4)
Date: July 24, 2005 11:56PM
Hi Dan,
Thankyou very much. Just completed my work. My site directed mutagenesis worked with your conditions. Though I had troubles initially it finally worked and I confirmed the results also. Once again thank you very much.
Re: Site directed mutagenesis
Posted by: rajabi (IP Hidden, Unregistered user, )
Date: August 7, 2005 04:50AM
I have plasmid which unfortunately occurs frameshift , of course framshift result from primer bad synthesis ( i recommended degenerate primer however both of nucleotide synthsis).
Plasmid= 4700 b.p. Insert =1200 b.p. Additional two nucleotide in first gene please how deletion two nucleotide (simple methods). Thank you very much for kind attention. rajabi
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