Amplify RANKL for cloning - (May/29/2007 )
Hi:
I try to amplify the RANKL (mouse) for cloning from Mc3t3-E1 cells but failed. I design the primer with extra REs( which is not include in the RANKL mRNA) : NheI and BamH I, but I can't amplify it at all. Anybody has some good suggestions for it?
I try to add additive to the PCR reaction: DMSO or BSA or fromamide , both of them doesn't work. There is 3000 bp band when i using 55 annealing tem. It is weired because the whole length of the RANKL is only 2400 bp. I tried annealing tem with 57, 58, 60. There is no band at all.
I guess: 1, the RANKL is low copy in the Mc3t3-E1 cell line;
2, The amplified product is GC rich: 55%;
Anyone has any suggestions?
Kind regards
H
Try touchdown PCR?
Check if your primer is ok for use. Make sure there's no likelihood of hairpin formation in your primer.
I try to amplify the RANKL (mouse) for cloning from Mc3t3-E1 cells but failed. I design the primer with extra REs( which is not include in the RANKL mRNA) : NheI and BamH I, but I can't amplify it at all. Anybody has some good suggestions for it?
I try to add additive to the PCR reaction: DMSO or BSA or fromamide , both of them doesn't work. There is 3000 bp band when i using 55 annealing tem. It is weired because the whole length of the RANKL is only 2400 bp. I tried annealing tem with 57, 58, 60. There is no band at all.
I guess: 1, the RANKL is low copy in the Mc3t3-E1 cell line;
2, The amplified product is GC rich: 55%;
Anyone has any suggestions?
Kind regards
H
Try a quick RT-PCR using primers that amplify a small portion of the gene to make sure it is actually there!
Also, scour the NCBI database to make sure you have the correct isoform/start codon etc. I been burned by this in the past.
http://www.ncbi.nlm.nih.gov/entrez/viewer....iew=gbwithparts
http://www.ncbi.nlm.nih.gov/entrez/viewer....&id=8843822
http://www.ncbi.nlm.nih.gov/entrez/viewer....iew=gbwithparts
Is it these?
http://www.ncbi.nlm.nih.gov/entrez/viewer....&id=2612924
Product should be around 900 bp!