To have a sure positive control for a PCR, I have ordered an oligo containing the cognate sequences of the primers (20 nt each) separated by 150 of random DNA; before and after the binding sites, I left five nt so the overall length is 200 nt.
When I ran the PCR, nothing was amplified.
Could it be that 5 nt upstream/downstream of the binding site are too few for the enzyme to work properly?
Also, could it be that having both cognate sequences on the same strand is a bad idea because the enzyme would collide releasing only small fragments?
When you say random bases do you mean just a single sequence of known bases or are you using Ns in your sequence?
The enzyme will work well even with no upstream bases but I worry that 20 bases is quite a low annealing temperature and if the unknown bases are random then randomly you may be getting self annealing or other secondary structure problems. It is also very easy to use far too much of a short sequence in a pcr. About 30,000 copies is commonly used and that is a tiny amount of a huge dilution of an oligo.
You could try running the pcr in presence of 5% DMSO or 1 Molar betaine to remove secondary structure problems and check the amount of template because 20 cycles of pcr is 1000000 times more product and you may be getting huge over amplification so the template reanneals with other template molecules and in the gel you get a smear but no band of amplimer and this smear may look like a failed reaction.
I do not think that any 2 sequences on a dna template will spoil a pcr just because they are on the same strand. It should be just the same as one longer sequence to be amplified
Thank you for the answer. For "random sequence" I meant that I used a plant genome sequence to ensure to be completely different from the bacterial cognate sites. But if the short leading and trailing sequences and the presence of the two cognate sites on the same strand is not a problem, then I think the secondary structure might beareit the problem, as you highlighted.
If it is a secondary structure problem then you can run a dmso gradient of zero t0 8% final concentration DMSO. Betaine can be used up to 2Molar but I usually found a final concentration of 1M works. You can make a 5M stock in water and add 1/5th of the reaction volume. Once you get a feel for what works they can be combined if needed for a cleaner product
good luck
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Pharmaceuticals
- Drugs