Hi,

both approaches should work in principle. You could also think of making it in two rounds, first to amplify the GOI and then to add the Overlap in a 2nd PCR reaction (in th eEnd a kind of nested PCR). In any case I would not go lower than 20 complementary bases as in my experience there was never an issue with the overhanging sequences

Sven

Sven Reister I see that sounds good. But these primers would be pretty huge. Two quick questions.

1) So the standard primer guideline of 18-30 bases considers only complementarity and not the final size of the primer?

2) I should be fine with any primer size as long as I have the requisite 18-30bp complementary sequence. Is there a theoretical upper limit as to how big primers can be (complementary region + end modifications) ?

Hello. Good time. Yes, we had a positive result in our experiment. It looks like a good sequence

Hello. Your talk is right. But only this pcr process responded to our testing and other methods did not respond

It seems to me that the gene is very interesting on Bacillus species. And nothing science is impossible

Hi,

I have used long Oligos (~75nts) regulary to introduce sequences in my plasmids e.g. when there was no appropriate restriction sites. the 18-30nt recommendation is only for the complementary part otherwise all the NGS workflows to add the seq primer sites and the barcodes would also have an issue. Therefore I think the approach should work as long as the complementatry part is fine

Sven

Sven Reister Thank you. The concern with length was becuause im amplyfying from genomic dna. But looks like it should be fine. Sudabeh Iraninasab Sir, l'm not sure I understand exactly what you are trying to say

Since I myself have been working on extraction of dna necroxic algae with three primers rbsl, col and lts-2. The pcr addition temperature was different for each, and dna extraction was successful only with the lts-2 primer. The only sequence that was transcribed in pcr. According to other studies, researchers can design a primer gene for the ncdi.

Excuse me to study the molecular identification of brown algae