PCR using a long (100nucleotides) and a short (20 nucleotides) primer. Problem: - (May/29/2006 )
I am trying to amplify a 1.8 kbp fragment using one long primer (100nucleotides) and a short (20 nucleotides) primer.
My protocol is:
30 to 90 ng template
100 pmols of each primer (Tm = 69C)
200 uM dNTP mix
1.5 mM MgSO4
10 u PfuTurboPolymerase
total vol 100 ul
I used a hot step 94C for 5 minutes without Pfu added. After adding the enzyme:
30 cycles of:
94C(30sec); 45C(1min); 72C(4min)
After these cycles I decreased the temp at 4C for 5 minutes
Result: no PCR product detected on 1% agarose gel!
It seems that the primers are self annealing and amplifying themselves since the band appears stronger on the gel after PCR. I tried different annealing temp from 45 to 64C but still no result!
Please give me some suggestions if you had one!!!
I would design a shorter primer, you shouldn't need a 100bp primer for any PCR, usually fragments of that size are used for Southerns or northerns.
I agree, go for a shorter primer, your 2 primers obviously have different annealing temperatures, thus no PCR product. Or if you really really need the long primer- for whatever reason, than make the 2nd one longer too. But shorter is better for PCR in general.
Not sure if your situation is as following: you have one 20mer primer (gene-specific/degenerate), but it is hard
for you to design another primer, So you try to make 100mer primer as a hybrid probe to fish out your target.
If it is truth, maybe there is other ways to do your PCR.
what i suggest :
do a 100µl PCR as follows.
In first 50µl, put template, dntp buffers enzyme..... and only 1primer (let say the 20ntd one)
do 10cycles with standard tm and extension time.
In "second" 50µl, prepare all but with only the 100ntd primer (don't put the 20ntd one.
Do a one step PCR (95°, 72°extension for appropriate time). Work with up to 10%DMSO in order to minimize the self anneal of priemr. Do 10 cycles.
Mix the 2 tubes. and do the rest of PCR over 20 25cycles.
Tha may help to obtain a product.
what is the reason for such a long PCR primer, surely at 20mer specificity is more than ample and the longer primer must be throwing off your annealing tempratures?
Thanks for suggestions! I designed a longer primer because I want to insert a signal sequence before my fragment of interest. So only the first 20 nucleotides are complementary to my template and that's why I have the same Tm (70C) for both primers. I am going to try what Fred33 suggested.
If you have other suggestions please let me know!!! Thank you!
A. too much template:
30 to 90 ng 1.8 KB template = 1.5-4.5E10 molecules of template if your template is the 1.8 KB fragment rather than genomic or plasmid DNA.
10 u PfuTurbo Polymerase = 2.5E11 molecules of enzyme, assuming the concentration of PfuTurbo is similar to AmpliTaq.
Four cycles using 1.5E10 molecules of template gets you to 2.4E11 molecules of template. When the molecules of enzyme and template are equal, you are in plateau phase, and amplification becomes linear rather than exponential.
B. A 100 nucleotide primer is likely to have secondary structure. Plug the sequence into the Oligo Analyzer program at www.idtdna.com. Check for structure, self-dimer and hetero-dimer with your 20 nuc primer.