I've performed a colony PCR with two unknown bacteria (in triplo). Lanes 6, 7 and 8 shows one bacteria with a nice outcome. The band that I expect is 1465 bp, because during the colony PCR, I use a 27 forward primer and a 1492 reverse primer. Lanes 3, 4 and 5 shows the other bacteria with also the expected outcome of 1465 bp, but there is also a band around 250 bp. Why is this band and what can I change in the PCR settings?
Hello,
it can be a parasitic amplification due to a hybridization of your primers at another place of the genome which generates this band.
You can try changing the hybridization temperature to avoid this unwanted hybridization and amplification.
Sofiane Benyamina Thank you for your answer. The annealing/hybridization temperature I used was 46,5 °C. What kind of temperature would you recommend? The bacteria is unknown, so I don't know what kind of hybridization temperature would be recommended for those.
it confirms what I suspected, your hybridization temperature is low, potentially your primer sequence is not rich in GC sequence or it is short, which favors parasitic hybridization.
Have you calculated the hybridization temperature according to the rule: hybridization temperature = Tm - 4°C.
A good primer should be between 18 and 23 nucleotides.
Sofiane Benyamina Thank you so much for your help. The forward primer is 20 nucleotides long and the reverse primer is 22 nucleotides long. The Tm of the forward primer is 46 °C and the Tm of the reverse primer is 47 °C. According to the calculated hybridization temperature, I have to use a temperature around 42-43 °C. But this is lower than de hybridization temperature I used before. Below you can see the primers I am using. Are you recommending the lower hybridzation according to the calculation, or should I go with a higher hybridization temperature?
Forward primer: 5` - AGA GTT TGA TCM TGG CTC AG - 3`
Reverse primer: 5` - TAC GGY TAC CTT GTT ACG ACT T - 3`
Hello,
I did not find the same Tm as you, try to redo the calculations of the Tm.
Tm = 2 (A+T) + 4 (G+C).
Normally it is higher than what you found as temperature.
Sofiane Benyamina You are right. When I calculate the Tm according to the formula, I get a the following temperatures:
Forward primer: Tm = 58-60 °C
Reverse primer: Tm = 62-64 °C
The Tm's of the primers are a little bit different. Which one should I use as guideline for the hybridization temperature?
I try to have the same Tm for the 2 primers, for example when designing the primers you could have removed the last G of the primer Forward primer: 5` - AGA GTT TGA TCM TGG CTC AG - 3`, this will have 19 nucleotides and it will not change anything to the hybridization and the 2 primers will have the same Tm. It's nothing you can use them but in this case of primer with different Tm, take the lowest temperature to calculate the hybridization temperature.
Sofiane Benyamina Hello, last week I've tested 10 different hybridization temperatures. All outcomes have the same extra band of 250 bp on the gel. Do you think that it is still the hybridization temperature or can I change something else about my protocol to avoid the extra band of 250 bp?
Hello,
If temperature annealing doesn't solve the problem, it means there is another region that strongly resembles your primers.
If you want to recover only the band of interest without taking the other band, cut out the corresponding band from the agarose gel and use a purification kit from a gel.
- Badges
- Science method