cDNA primer help - (Nov/15/2008 )
Hi everyone, I have done my search on this in these forums and it seems that still after 4 hours of reading I'm still a bit confused on this matter.
I did the Blast on the Mus musculus amyloid beta gene and in order for me to design the primer I need to click the CDS button to align the sequence from the staring codon with ATG. So now I need to design the primer! My question is, when designing a primer, is this cDNA sequence going from 5'--->3' and therefore in order to design the primer I have to base pair the primer from the 3' end so that 5'starts with the primer I'm designing???
Also, the primer must have a Tm of at least 68-72 degrees and gc% of 50%.
For example: I need to highlight the primer positions and write the primer sequences and Tms for the primers which I'm going to use for the PCR.
atggcggcca ccgggaccgc ggccgctgcg gccaccggca agcttcttgt cctgctgctg
ctcgggctca cggcgcccgc tgcggctctg gctggctaca tagaggctct tgcagccaat
gctggaacag gcttcgctgt tgctgagcct cagattgcga tgttctgtgg gaagctgaat
atgcatgtga acattcagac tggcaaatgg gagcctgacc caacaggcac caagagctgc
cttggaacaa aggaggaggt tcttcagtac tgccaggaga tatatccaga gctgcagatc
acaaatgtga tggaagcaaa ccagccagtc aatattgata gttggtgccg aagggacaaa
aggcagtgca agagtcacat tgttatacca ttcaagtgtc ttgtgggtga atttgtaagt
gatgtcctgc tagttccaga taactgccag tttttccacc aagagcggat ggaggtgtgt
gagaagcacc agcgctggca cacgttagtc aaggaggcat gtctgactga ggggctgacc
ttatatagct atggcatgct gctgccctgc ggggtagacc agttccatgg caccgagtat
gtgtgctgcc ctcagacaaa gactgttgac tcggactcga ctatgtccaa agaagaggag
ccagaagagc gtcacctgaa caagatgcag aaccatggtt atgaaaaccc aacctacaaa
tacctggagc agatgcagat ttaa
Do I need to start with red or the blue?
would the blue be the lower primer and therefore it would be TTAA ATCTGCATCA GCTCCA
Tm= 56 degrees.
I don't know if I'm doing this right.
I'm open for any suggestions
I would appreciate your help..
Sequences you see are ALWAYS written 5' to 3'.
I can't see any colours that you have put in your sequence, so I don't know what the "blue and red" you refer to are highlighting. You mainly need to look for a sequence with 50% G/C content as the TM is dependent on the GC content mostly
You should know this equation by now:
TM=4*(G/C) + 2*(A/T)
Don't forget that your reverse primer is the reverse complement of the sequence you are looking at.