DNA quantification and probe selection - (Dec/22/2005 )
Hi,
in fact i earlier posted doubt on ish,but no replies came.I hope somebody may answer this post.
For ish,i have to use a probe,how anybody selects whethert the probe should be dna or oligomer?how this Sequence of DNA is produced for labeling?
Two methods i came to know
1.after a solution pcr,product is eluted from the gel and labelled?
2.sequence has to be found out using softwares,and then can be synthesized?
my product is 123bp, so what am i to do?
before labelling ,the DNA shud be quantitated,what is the procedure for that?
Waiting for your suggestion,Thank you.
-niveda-
QUOTE (niveda @ Dec 23 2005, 12:18 AM)
Hi,
in fact i earlier posted doubt on ish,but no replies came.I hope somebody may answer this post.
For ish,i have to use a probe,how anybody selects whethert the probe should be dna or oligomer?how this Sequence of DNA is produced for labeling?
Two methods i came to know
1.after a solution pcr,product is eluted from the gel and labelled?
2.sequence has to be found out using softwares,and then can be synthesized?
my product is 123bp, so what am i to do?
before labelling ,the DNA shud be quantitated,what is the procedure for that?
Waiting for your suggestion,Thank you.
in fact i earlier posted doubt on ish,but no replies came.I hope somebody may answer this post.
For ish,i have to use a probe,how anybody selects whethert the probe should be dna or oligomer?how this Sequence of DNA is produced for labeling?
Two methods i came to know
1.after a solution pcr,product is eluted from the gel and labelled?
2.sequence has to be found out using softwares,and then can be synthesized?
my product is 123bp, so what am i to do?
before labelling ,the DNA shud be quantitated,what is the procedure for that?
Waiting for your suggestion,Thank you.
if u are talking about Insitu hybridization probe
u can make an antisense riboprobe which is specific for your mRNA
first PCR amplify the segment from a cDNA using primers which add restritcion sites which u can use to clone in a vector such as pGEM-3z(promega)
this vector has SP6 and T7 promoters which can be used to make sense or antisense riboprobe byin vitro transcription ..u can use radiolabelled NTPs in the reactions or digoxin labelled (non-radioactive) method
hope that helps
-Watson-