C121W mutation is relate to GEFS+. It's screening can be done by amplifying exon 3 of SCN1B gene.
Exon 3 of SCN1B gene is amplified by following primers:
5'-CCTTCCCCTCCCTGGCTAC-3' (Forward)
5'-AAGAGCAGGCGGTAGACGCG-3' (Reverse)
with 30 cycles and an annealing temperature of 59C (degree celcius). The concentration of various other components of the PCR master mix has not been mentioned anywhere.
In such a case, how should the master mix be prepared?
Dear Shrestha,
There are many factors to be taken care to prepare a master mix and also it is very easy.
Your question is clear, for the amplification of that particular exon, you need to have DNA template with 10 ng/ul concentration. I think your intention is to sequence this amplicon for mutation detection, So, you need to minimize the amount of dNTPs, Taq DNA Polymerase, Primers and so on, because all these will affect the quality of the DNA sequencing. I donno which concentration of working primer you are using. Use 5 picomol of primers, both the forward and the reverse primers. You can use 0.5μl of primers; excess use of primers will result in primer dimmers also. I am not sure which PCR mix you are using, whatever, try to optimize the minimum amount of reagents. The below given case report might be useful for your for further clarifications, if any, Good luck
http://www.smj.org.sa/PDFFiles/Dec09/02Anovel20091022.pdf
Hi.Dear Shrestha
There are several kinds of ready to use Master Mix that you can use them readily such as Amplicon Master Mix which is also benefited from Hot-start PCR
in situation that you cannot optimize PCR reaction simply, in my opinion, you can use these master mixes. However, some of them are no cheap.
before using, please pay attention to the concentration of master mix for example 2X or 5X. Due to the concentration the amount of master mix will be changed.
I always use 5X master mix because in a 25 micoliter reaction I need 5 microliter of master mix where all PCR materials already have been provided.
Good Luck
The master mix usually is determines by the enzyme you are using. What enzyme and from what manufacturer are you using? The protocol provided from the company is the best place to start.
Then use a piece of software to help determine annealing temp. I find the one below from IDT very good. You will see that it predicts the Tm of your forward primer to be at 59C, so an annealing temp of 59 might be a bit high and worth dropping a bit.
http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
I also typically run my PCRs for 35 cycles.
@Stephen Doyle: I looked at the oligoanalyser tool from the link you suggested. It was really good.
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