Hello.
I have a problem on pcr with overhang primer
The total length of primer is 90 bp and annealing region is 25bp
GC content% of annealing site is 40~45%
I want to extend the gene about 60 bp at the end of 3' and 5' by pcr reaction.
and total 3 times of pcr reaction was conducted to extend 60*3=180bp at the end of 3' and 5'
The result of first time of pcr was good (fig left) the band was clear as I wanted
In second time of pcr, the pcr product was shown as expected size but very faint.
In the third time of pcr, using a template from the product of second time,
no band is as a result TT
Question)
1) I want to know how to make pcr reaction clear? Especially with overhang primer.
2) As picture shown, the DNA ladder was unclear.... I don't know why it happened. Sometimes the DNA ladder was not shown.... If you know the reason of it plz help.
Sincerely thank you
When you say that the PCR
When you say that the PCR doesn't work, does this mean you get nothing on the gel, a smear, or the wrong sized band?
How many G/C and A/T do you have annealing in the first round of the PCR for each primer?
Knowing this I would consult an annealing table I have (because the 4 and 2 rule is too unreliable). It gives the temperature at which 50% of the primer binding sites will be bound so I'd drop the temperature about 1 degree below this.
Also you might think about using a slower than normal temperature ramp from the 95 degrees to the annealing temperature just calculated.
If you have a PCR machine with a gradient block then it's probably a good idea to run a number of samples (usually up to 12) with annealing temperature varying between Tm-5 degrees to Tm+15 degrees - then you'll see when you get the most efficient annealing.
Remember that in subsequent rounds of PCR, the longer oligo will be annealing before the shorter one since the template will be derived from an earlier PCR product that includes the long oligo's complementary sequence.
If you post the (1st round annealing) GC and AT content (eg. 18:12 respectively) for each primer I'll check it on my table and post what I get
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