will site directed mutagenesis work for 8kb construct? - (Jul/10/2008 )
Hi,
I have a situatioin here. I have cloned a total of five genes in series (together ~3kb) using various enzyme sites in a mammelian expression vector pCAGGS (~4.7kb), i.e. a construct of ~8kb. Now I need to change some of the amino acids from the construct. Is it possible to use site directed mutagenesis to do this? Is there any other alternative that I can use? If anyone has encountered similar situation, Please kindly guide me.
Thanks
-nageshipper-
QUOTE (nageshipper @ Jul 10 2008, 07:00 PM)
Hi,
I have a situatioin here. I have cloned a total of five genes in series (together ~3kb) using various enzyme sites in a mammelian expression vector pCAGGS (~4.7kb), i.e. a construct of ~8kb. Now I need to change some of the amino acids from the construct. Is it possible to use site directed mutagenesis to do this? Is there any other alternative that I can use? If anyone has encountered similar situation, Please kindly guide me.
Thanks
I have a situatioin here. I have cloned a total of five genes in series (together ~3kb) using various enzyme sites in a mammelian expression vector pCAGGS (~4.7kb), i.e. a construct of ~8kb. Now I need to change some of the amino acids from the construct. Is it possible to use site directed mutagenesis to do this? Is there any other alternative that I can use? If anyone has encountered similar situation, Please kindly guide me.
Thanks
Sure it will work.
See some links here for further guidance.
http://search.vadlo.com/b/q?sn=158621799&a...s+kb+&rel=0
..
-cellcounter-
QUOTE (cellcounter @ Jul 11 2008, 02:43 PM)
QUOTE (nageshipper @ Jul 10 2008, 07:00 PM)
Hi,
I have a situatioin here. I have cloned a total of five genes in series (together ~3kb) using various enzyme sites in a mammelian expression vector pCAGGS (~4.7kb), i.e. a construct of ~8kb. Now I need to change some of the amino acids from the construct. Is it possible to use site directed mutagenesis to do this? Is there any other alternative that I can use? If anyone has encountered similar situation, Please kindly guide me.
Thanks
I have a situatioin here. I have cloned a total of five genes in series (together ~3kb) using various enzyme sites in a mammelian expression vector pCAGGS (~4.7kb), i.e. a construct of ~8kb. Now I need to change some of the amino acids from the construct. Is it possible to use site directed mutagenesis to do this? Is there any other alternative that I can use? If anyone has encountered similar situation, Please kindly guide me.
Thanks
Sure it will work.
See some links here for further guidance.
http://search.vadlo.com/b/q?sn=158621799&a...s+kb+&rel=0
..
Thank you Cellcounter! I really appreciate this.
-nageshipper-
QUOTE (nageshipper @ Jul 10 2008, 06:00 PM)
Hi,
I have a situatioin here. I have cloned a total of five genes in series (together ~3kb) using various enzyme sites in a mammelian expression vector pCAGGS (~4.7kb), i.e. a construct of ~8kb. Now I need to change some of the amino acids from the construct. Is it possible to use site directed mutagenesis to do this? Is there any other alternative that I can use? If anyone has encountered similar situation, Please kindly guide me.
Thanks
I have a situatioin here. I have cloned a total of five genes in series (together ~3kb) using various enzyme sites in a mammelian expression vector pCAGGS (~4.7kb), i.e. a construct of ~8kb. Now I need to change some of the amino acids from the construct. Is it possible to use site directed mutagenesis to do this? Is there any other alternative that I can use? If anyone has encountered similar situation, Please kindly guide me.
Thanks
Hi
I have used site directed mutagenesis a number of times on targets larger than 12kb with absolutely no problems - I would recomend Pfu turbo from Stratagene. FYI Stratagene also do some sort of kit that allows you to perform multiple mutagenesis on a single target - I have never tried it, but it looks quite interesting...
-jenjohn-
Thank you john~!
-nageshipper-