HOW to clone some kinases? - clone CDK2,CDK3,MAPK14 from HeLa (Oct/24/2008 )
Hi, i want to clone some kinases from HeLa, for example ,CDK2,CDK3,MAPK14.
i have search the Human Proteinpedia, and these kinase have the expression in HeLa.
so, i do the RT-PCR, but nothing except of primers.
of course, these kinase mRNAs is low , I do another pcr in which the previous pcr products as templates and the reaction conditions have been optimized, but nothing still.
anyone could kindly give me some suggestion ?
Hi
Sorry I have no idea about how you should
solve your problem but I need your help
Hey guys,
Can someone tell me how to design a RT-PCR primer?
I am the first one(first time) to do it in my lab.
I got a couple of informations from the net but some are
just to difficult to understand for a beginner like me.
What is the most important factor (Max score, Total score
,E-value...) to look when you blast the amplicon sequence
(or the primers)against genome database.
Thank you for your help.
Sorry I have no idea about how you should
solve your problem but I need your help
Hey guys,
Can someone tell me how to design a RT-PCR primer?
I am the first one(first time) to do it in my lab.
I got a couple of informations from the net but some are
just to difficult to understand for a beginner like me.
What is the most important factor (Max score, Total score
,E-value...) to look when you blast the amplicon sequence
(or the primers)against genome database.
Thank you for your help.
most RT-PCR kit just produce first cDNA, the primer used is oliglT or random primers which the kit supplyed
next, i often use primer premier5 to design the specific primers to amplify the target genes.
you can load the primer -BLAST to blast the amplicon sequence
[b]http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome
good luck