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Hello, all! We think that we have problems with fragment library
preparation for SOLiD 4.0. We worked using standard protocol, but
created library didn't cover whole reference genome. Local FAS, said
that our problem - is reamplification, but we don't think so.
Fragment library preparation
Fragment library preparation
Starting probe volume: 75 mkl. DNA concentration: 4 mg
Shearing by Covaris: standard program from manual for SOLiD 4 Library preparation
End-repear - 30 min (25 degrees)
After column purification concentration (Quibit – BR) - 60 ng/mkl in 100 mkl
We divided sample on two probes with same DNA concentration
After that we worked with one of this probes
Calculation volume of P1- P2 –adapters - 16, 5 mkl
Ligation – 15 min (25 degrees)
Purification on columns
Size-select (2% agarose)
Purification on columns after size-select
DNA-concentration (Quibit – BR) - 72,2 ng/mkl in 50 mkl (3.6 mg)
LibraryPCR (standard conditions, 3 cycles)
Purification on columns, DNA-concentration (Quibit – BR) - 30 ng/mkl in 50 mkl (1.5 mg)
preparation for SOLiD 4.0. We worked using standard protocol, but
created library didn't cover whole reference genome. Local FAS, said
that our problem - is reamplification, but we don't think so.
Fragment library preparation
Fragment library preparation
Starting probe volume: 75 mkl. DNA concentration: 4 mg
Shearing by Covaris: standard program from manual for SOLiD 4 Library preparation
End-repear - 30 min (25 degrees)
After column purification concentration (Quibit – BR) - 60 ng/mkl in 100 mkl
We divided sample on two probes with same DNA concentration
After that we worked with one of this probes
Calculation volume of P1- P2 –adapters - 16, 5 mkl
Ligation – 15 min (25 degrees)
Purification on columns
Size-select (2% agarose)
Purification on columns after size-select
DNA-concentration (Quibit – BR) - 72,2 ng/mkl in 50 mkl (3.6 mg)
LibraryPCR (standard conditions, 3 cycles)
Purification on columns, DNA-concentration (Quibit – BR) - 30 ng/mkl in 50 mkl (1.5 mg)