Commercial qPCR advantage?

11-17-2012, 06:26 PM

While reading on quantifying libraries with qPCR, people mentioned using commercial kits quite often...

What is the advantage of using a commercial qPCR kit (for libraries), e.g. Kapa, instead of a "homemade" mix with in-house qPCR reagents and ordered primers?

yaximik · 11-18-2012, 07:24 AM

None, if one wants to spend time creating a standard and finding a sensitive reliable SYBR mastermix that satisfies the expected range. Standards of different sizes can be simply cloned from one of sequencing libraries and reamplified from selected plasmids as needed.There are a whole lot of mastermixes out there in $200-300 range or even less for 5 ml, which is enough for 250-500 assays. KAPA is not the best one in sensitivity as they claim, as it contains quite a lot of SYBR that inhibits reaction and it consistently produces false signal in negative control after cycle 35.

11-20-2012, 10:16 AM

Thank you for the reply. I was just wondering as I tried it for the first time with our Platinum SYBR green and it worked out well. Did not see any false positives. Nonetheless everybody was mentioning KAPA and it is even suggested in Illuminas tech note, oh well...

yaximik · 11-20-2012, 11:24 AM

I did not say KAPA is bad, it is certainly OK otherwise it would not be recommended. I just thought that paying $500+ for it when something of a similar or even better performance can be found for twice less does not seem smart...

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11-17-2012, 06:26 PM	#1
lorendarith Guest Posts: n/a	Commercial qPCR advantage? While reading on quantifying libraries with qPCR, people mentioned using commercial kits quite often... What is the advantage of using a commercial qPCR kit (for libraries), e.g. Kapa, instead of a "homemade" mix with in-house qPCR reagents and ordered primers?

11-18-2012, 07:24 AM	#2
yaximik Senior Member Location: Oregon Join Date: Apr 2011 Posts: 205	None, if one wants to spend time creating a standard and finding a sensitive reliable SYBR mastermix that satisfies the expected range. Standards of different sizes can be simply cloned from one of sequencing libraries and reamplified from selected plasmids as needed.There are a whole lot of mastermixes out there in $200-300 range or even less for 5 ml, which is enough for 250-500 assays. KAPA is not the best one in sensitivity as they claim, as it contains quite a lot of SYBR that inhibits reaction and it consistently produces false signal in negative control after cycle 35.

11-20-2012, 10:16 AM	#3
lorendarith Guest Posts: n/a	Thank you for the reply. I was just wondering as I tried it for the first time with our Platinum SYBR green and it worked out well. Did not see any false positives. Nonetheless everybody was mentioning KAPA and it is even suggested in Illuminas tech note, oh well...

11-20-2012, 11:24 AM	#4
yaximik Senior Member Location: Oregon Join Date: Apr 2011 Posts: 205	I did not say KAPA is bad, it is certainly OK otherwise it would not be recommended. I just thought that paying $500+ for it when something of a similar or even better performance can be found for twice less does not seem smart...