So I know in molecular biology that the choice of primers is important. Some primers like to form primer dimers or trimers, especially if length of the primer in nucleotides is large. I have been trying to polymerase chain reaction (PCR) amplify a gene which is 1344 base pairs in size. I used a forward primer (78.2°C melting temperature, 16739 Molecular weight) and a reverse primer (70.2°C melting temperature and 13540 molecular weight). The forward primer had the EcorI restriction site, and the reverse primer had the HindIII restriction site, the sequence of each primer starting with those 6 nucleotide cut sites.
I used the Phire II polymerase and ran the thermocycler at the conditions:
98°C (30 seconds) -> {98°C , 5sec -> (62 - 68°C, 5 sec) -> 72°C, 1 min}(repeat 39 times) -> 72°C (1 min) -> 12°C, infinite hold.
For my PCR reaction, I used 5 uL of the forward primer, 5 uL of the reverse primer, 4 uL of the 5x Phire Buffer, 0.4 uL of dNTP, 1 uL of my template DNA, 0.4 uL of Phire II polymerase, 4.2 uL of H2O. All this in triplicate.
On my gel reading right to left is a 1 kb DNA ladder followed by 3 replicates of my PCR amplified gene. On this gel I skipped lanes. My gel is 1% Agarose. (By the way, the gel was stained with ethidium bromide with an ultraviolet light shining at 365 nanometers.)
I extracted (using a thermoscientific genejet gel extraction kit) a result that looked kind of like this gel before with the same gene:
127.8 ng/uL concentration and 1.85 is the ratio of absorbance at 260 to 280 nanometers.
I thought that because I had such bright bands that I must have had the DNA but when I tried to transform this DNA insert into a plasmid, the ligation didn't work, but the empty plasmid transformed fine. So there must be something wrong with my insert which is this.
My supervisor said that it looks like I extracted primer dimers? That the DNA was bands on the gel are like 300 base pairs when they should be 1344 base pairs in size. Do you think that is true? What do you think the size of my bands are?
Do you think that there is an issue with my thermocycler settings that is not allowing the primers to anneal or maybe the Phire II polymerase is not working up to par with this and I should use Phusion. (I have been using Phusion but we need to order some more.)
Why do they call the DNA ladder 1 kb DNA ladder if the size ranges from 500 base pairs, to 10 kb? https://www.neb.com/products/n3232-1-kb-dna-ladder#Protocols%20&%20Manuals. I am more experienced at computational biology and education biology so please help.
Thanks!
They might or might not be the primer dimers. Try to run the gel longer so that the ladder's bands are more separated. For 1kb-ladder the bands do look like less than 500bp (it's called a 1kb-ladder because the distance b/w bands is 1kb starting with 2kb-band). It also looks like the concentration of the DNA template is very high which might explains the brightness of the bands. Try to dilute the DNA template for the PCR mixture. The region of 1344bp shouldn't be a problem for the amplification if the primers work. I've also noticed that annealing temperature for your primers is unusually high.
Hope that helps!
Hi,
To me it also looks like you may have primer dimer. You said you used 5ul each primer. But what was the concentration you used. You may try using 2uM each primer concentration. You used 72C for 1min for DNA synthesis. But your product is around 1350bp, you may need to increase the synthesis time to 1.5 min. Usually 1kb DNA synthesis need 1 min. And annealing time is also seem very low 5 Sec, try to increase it to 30 Sec. Thank you.
Hi Adron,
100 times primer dilution sounds ok to me. May be you can pay attention to anneal and synthesis time. You can do a gradient PCR to find out perfect anneal temperature. Thank you!
Adron, the primers I work with for DNA barcoding typically have annealing temperature 45-60 C. I agree with MdRiaj Mahamud that a gradient PCR will help to find the right annealing temp. Also, the specification sheet for the primers should have approx. range for the annealing temp (although it's not perfect most of the time).
Hey Alina, thanks for the suggestion. I am making progress. I found that adding DMSO helps with decreasing the effective melting temperature of my primers and thus makes annealing of my primers more efficient at an annealing temperature of about 66 to 68°C. I wasn't able to obtain any desirable bands without DMSO, but it is not enough to extract yet. It looks like I will have to order new primers and transform more template into E. coli cells. So that would give me a higher concentration of DNA template which may help the primers do their job.
I see the primer dimers and nucleotides in the bright bands. Template should not be that small, so I don't think it is template showing bright bands. Every enzyme is different in physical properties. For Phire, you only need about 30 seconds of polymerization and 1 minute is too long. It may help lower the annealing temperature and increase annealing time to 30 sec. If you added enzyme site sequence in the primers that are not in the template, you should not consider that extra sequence in the primers for calculating Tm. That's why you should lower the annealing temperature, in my opinion. Having too many cycle can generate non-specific bands, but in your case it is not an issue.
Adron Ung, a back of the envelope calculation tells that your 16739 MW primer is something around 54 bases long, and the other around 43 bases. That is rather long and may be a reason for the lack of a proper annealing. The longer the complementary region, the longer it'll need for annealing (and denaturation), and the higher chance that it can bind to things that are not completely complementary if your annealing temperature is too low. To me, your 5 sec denaturation (98 C) and annealing (62-68 C) steps seems a bit short. In general, a complementary region of 10-20 bp is sufficient... that also can keep the melting temperature and thus annealing temperature low.
You may want to try:
Thermocycling settings
- longer initial denaturation step (2 min?) and cycle denaturation (0.5-1 min) and annealing steps (0.5-1 min).
- As mentioned by Md Riaj Mahamud & Alina Avanesyan , a gradient PCR for the annealing step can help you find the right temperature.
- It is also quite common for such kind of PCR to first run 2-3 cycles at an annealing temperature for the Tm of ONLY the complementary region, and then complete the rest of the cycles with a slightly higher annealing temperature that matches the Tm of the full primer sequences.
Redesign a new primer pair
- shorter complementary sequences say ~15 bases
- add in the restriction sites as usual,
- plus 2-3 addition nucleotides (G/Cs) 5' to the restriction sequence (which usually facilitate cutting by the enzymes in subsequent step)
For general molecular cloning techniques, I recommend reading the textbook classic "Gene Cloning & DNA Analysis" by T.A. Brown.
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