Hey

You should use the output directly (so you should order CCCCCGGGGCGATTGAT).

You can see that that sequence matches your template on the minus strand, which is what you want, since you want the reaction to extend in the reverse direction.

it is a standard that you always write the sequence in the 5' to 3' direction (and the company where you are ordering will ask for them to be written this way), which is why the BLAST primer design programme already gives it in that format.

I would check for the quality of your reverse primer though, since you have many Cs and Gs close together, which could give you secundary structure formation problems.

Cheers,

Hey

You should use the output directly (so you should order CCCCCGGGGCGATTGAT).

You can see that that sequence matches your template on the minus strand, which is what you want, since you want the reaction to extend in the reverse direction.

it is a standard that you always write the sequence in the 5' to 3' direction (and the company where you are ordering will ask for them to be written this way), which is why the BLAST primer design programme already gives it in that format.

I would check for the quality of your reverse primer though, since you have many Cs and Gs close together, which could give you secundary structure formation problems.

Cheers,

I agree with Nuno Leitao. I add that the reverse primer is not good in this case as it would result in self dimer. I suggest that its better to select an alternative reverse primer that does not have stretches of identical nucleotides. The general rules is that any nucleotide should not be repeated more than 3 times in a prime. In this reverse primer the situation is worst as you have longer and adjacent repeats of G,s and C,s

I don't know what use those primers are for, but as stated above the R primer is of a doubtful "quality". I would recommend to use tools like primer3 (http://fokker.wi.mit.edu/primer3/input.htm) or BLAST primer designing tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) to be sure to use primers which are both specific of your target and with no (or the less possible) internal problems (self-hybridization, dimers, 3'end complementarity or hairpin / secondary structures formation). Primer3 gives more details about quality of your primers / primer pair, you should try to minimize any quality parameter.

As said above, primer programs usually give primer sequence from 5' to 3', as you should order them. Good luck.

It is usually a good idea to let people know what you are trying to amplify, as it allows a reader to design alternatives without having to search for the intended target. Using PrimerBlast, it looks as though you are intending to amplify U13369.1 Human ribosomal DNA complete repeating unit. The problem here is the extreme GC content of the sequence

As said before, your reverse primer breaks every rule of every PCR manual, with a run of 11 GCs making it virtually certain you will get nonspecific amplification and problems with annealing.

I have used Beacon Designer (Premier Biosoft) to design a couple of sets primers and they are attached in the excel file for your further analysis. The third primer set uses the forward primer you have already described in your message. Good luck

Hi, I totally agree with Stephen. The reverse primer forms a hairpin-loop with a melting temperature of 37.5°C, and the forward primer with 23.3°C. If you use a proof-reading polymerase with exonuclease activity and no hotstart protocol, the forward primer will be elongated at the 3'-end of the hairpin loop, which will contribute to false-priming...