Bisulfte PCR and sequencing PCR and Primer Notes - Tips and hints on PCR for bisulfite converted DNA and bisulfite primer (Jan/08/2007 )
Dear nick:
After reading your primer notes,i am still confused when i design my nested pcr primer of BSP,can you help me？waiting for your reply.thank you!!!
xyr05
xyr05,
here are the primers I have picked, they are heminested set, so the first round use 1F and 1R and for the second round use 2F and 1R.
You need to calculate the Tm with your favourite calculator and stick with it, should be around 60C can go down to 50C but make sure the primers don't have a Tm difference of greater than 2C. Change the Tm by adjusting the length from the 5' end of each primer.
good luck!
Nick
edit: forgot the file!
edit2: try again!
Dear nick:
Thank you for your help again, if you have time,please upload the file to this thread or send it to my Email (xyr-1982@163.com) . looking forward your reply!
Best regards, Yirui Xie
Hi:
I am a student of Inner Mongolia Agriculture University of China .Now I will do experiment about DNA methylation area ,but I have no any experience at this area and my classmate don’t know this.Now I don’t know who I can ask. So when I see this web today ,I am very glad. At the beginning of experiment, I face some problem that I want to study the DNA methylation of abnormal vertebrae of sheep ,but I don’t know which gene control vertebrae and whether it is methylation region ,I think if I can’t find the gene ,I can’t design the primer.and PCR .I don’t know whether I am right ?if I have some false, please correct me ! I am so sorry that i know nothing abt these..
i would be very greatfull if you could help me out or refer me some papers or books to read about this..
thank you very much!
Hi yonggi 1,
I guess you are right, if you don't know the gene, then methylation won't be the first step. Maybe you can do some literature search for possible candidate genes involved in your topic?
I guess you are right, if you don't know the gene, then methylation won't be the first step. Maybe you can do some literature search for possible candidate genes involved in your topic?
Thank you very much for your reply! I am really very happy to see your answer! Now I want to find the gene controling vertebrae variation,i don't how to find,so i look for many paper and a paper say hoxc-8 can effect vertebrae develop.i don't know whether it is suitable. I want to try it, but i can't design the primer.i am confused but i will try my best to do the experiment and hope you can give me some advice ,thank you !
Hi yonggi, have alook at the pinned topic How to find the promoter of a gene - there is a step by step protocol how to find the promoter and how to select primers.
Best luck!
K.
Hi krümelmonster :
My English is not good,I don't know how to express my appreciation better,in a word,Thank you very much! Hope you have a good luck too!
Hi mrthylnick
Thanks for all that useful info. I'm a third year PhD student and I'm trying to analyze the methylation status of the promoter of a gene.
I have succeeded in amplifying the region of interest after bisulfite conversion but I am having trouble cloning this product.
I have been using the pCR2.1-TOPO vector from Invitrogen TA cloning kit but after sequencing 20 clones only one had the complete 327bp PCR product present. The rest of them had approx 200bp of the product with the 3' end missing. This keeps happening and I can't figure out why.
Any suggestions would be really appreciated.
AOG
hi AOG,
i suspect the bisulfite conversion has created a TOPO site and u are getting aberant recombination.
TOPO is a great way of cloning, we use pGEMT but are switiching to TOPO and have found similar thing was occurring.
can't be rectified unless you use older sequencing vectors such as pGEMT
N