Hi There,
I have recently encountered a problem about library quantification that has hindered my project for months. And i have browsed through all the questions here, it seems this has never happened to anyone before. I really hope if anyone knows how to solve this. Many thanks
After i generated my library, i did:
1: qubit to measure concentration, worked fine, concentration between 80-300 ng/ul
2: did a TA clone and sent 20 clones for sequencing to look at the accuracy of primer sequence, more than 80% are 100% accurate.
3: Did a regular PCR using illumina primer set (p5/p7) under a serial of anneal temperature (54-70), got very strong product at the correct size.
The problem comes when i use KAPA qPCR kit, i used a 4pM and 2pM (adjusted according to qubit result) as template, the result always showed less than 1% of the library was recovered. I should also mention that i included a control sample from someone else when i did all the above experiments. They worked the same in the first three, but differ at qPCR, with my sample came out much much lower than the control.
This has been driving me crazy, please help.
I have recently encountered a problem about library quantification that has hindered my project for months. And i have browsed through all the questions here, it seems this has never happened to anyone before. I really hope if anyone knows how to solve this. Many thanks
After i generated my library, i did:
1: qubit to measure concentration, worked fine, concentration between 80-300 ng/ul
2: did a TA clone and sent 20 clones for sequencing to look at the accuracy of primer sequence, more than 80% are 100% accurate.
3: Did a regular PCR using illumina primer set (p5/p7) under a serial of anneal temperature (54-70), got very strong product at the correct size.
The problem comes when i use KAPA qPCR kit, i used a 4pM and 2pM (adjusted according to qubit result) as template, the result always showed less than 1% of the library was recovered. I should also mention that i included a control sample from someone else when i did all the above experiments. They worked the same in the first three, but differ at qPCR, with my sample came out much much lower than the control.
This has been driving me crazy, please help.
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