I am doing PCR to add a (GGGGS)4 linker at one end of a gene. After amplifying the gene with suitable forward and reverse primer, I got an excellent band on the gel, eluted it from the gel, and checked the agarose gel again. I used the same (the eluted sample) as the temple to amplify with an old forward and new reverse primer having 10 bases of linker sequence at their 5' end. I got an excellent band on gel again, eluted the same, and checked on the agarose gel. But this time when I used elutes sample for PCR with the same primer set, this time no band was observed on the gel. I repeated the PCR with the dilution of the DNA temple, changed the PCR master mix (Takara), and added DMSO to the master mix.
How much amplified template did you use for the second amplification and how many pcr cycles did you use?
You need about 30000 copies of the template dna which will be a huge dilution of the dna. Every 10 cycles of pcr is 1000 times more product so very few cycles are needed otherwise the pcr product anneals to other product molecules and you just get a smear of amplified dna along the full length of the gel and not a single band of re amplified material. I would expect dilutions of about 1:1000000 and 26 cycles of pcr to get a clean product but you are better off calculating the dilution to give 30,000 copies of the template amplimer
Adding dmso is a good idea to solve secondary structure problems and to minimise G quadruplex formation in your primers but be careful if you are using highGC buffer from Takara it is probably dmso and betaine so using high GC buffer and additional dmso may take the dmso concentration above 10% which will deactivate the enzyme
Thank you so much, Paul Rutland, for your valuable suggestion. I am trying the same, hope I will get a positive result.
Here are the things, the concentration of the temple DNA of the first PCR product was 20ng/ul (Nanodrop), and I used 1 ul for the second PCR in a 20 ul reaction. After gel elution the concentration of DNA was 21ng/ul, again I used 1 ul and 1ul of each 20, 40, and 60 times diluted samples. I got no band after 25 cycles but the smear along the gel.
I think that those dilutions are not enough,What length is your amplimer please. Meanwhile if it is not too late try dilutions of 1/1000 and 1/10,000
If we apply the equation
copies= ng dna x 6.022x10exp23/(length x 10exp9 x 660 and I assume the length of your dna amplimer is 5000 bases then we get approximately 21 x 6 x 10exp23/5000 x 10exp9 x 660 which is very approximately 4x 10exp9 copies in your 1ul of 21ng dna.
You may need to dilute by a factor of 4x 10exp9/30,000 so run a few dilutions down to 1:100,000 in your pcr and maybe 25 cycles
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