FaqI (BsmFI) (2 U/µL)

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	Genomic DNA was extracted as a template from each embryo following the HotSHOT protocol as described by Meeker et al.4 when the desired assays were done. alas1smu350/smu350 and alas1Δ2/Δ2 mutants were genotyped by PCR followed by FaqI (Thermo Scientific, San Jose, CA, USA) and BsrI (New England Biolabs, Ipswich, MA, USA) digestion, respectively. The alas1smu350/smu350 PCR products were digested by FaqI into two fragments of 57 bp and 85 bp, the alas1Δ2/Δ2 PCR products were digested by BsrI into two fragments of 43 bp and 97 bp, whereas the WT PCR products were resistant to the digestion.

Protocol tips

Genomic DNA was extracted as a template from each embryo following the HotSHOT protocol as described by Meeker et al.4 when the desired assays were done. alas1smu350/smu350 and alas1Δ2/Δ2 mutants were genotyped by PCR followed by FaqI (Thermo Scientific, San Jose, CA, USA) and BsrI (New England Biolabs, Ipswich, MA, USA) digestion, respectively. The alas1smu350/smu350 PCR products were digested by FaqI into two fragments of 57 bp and 85 bp, the alas1Δ2/Δ2 PCR products were digested by BsrI into two fragments of 43 bp and 97 bp, whereas the WT PCR products were resistant to the digestion.

Publication protocol

"Genomic DNA was extracted as a template from each embryo following
the HotSHOT protocol as described by Meeker et al.4
when the desired
assays were done. alas1smu350/smu350 and alas1Δ2/Δ2 mutants were genotyped
by PCR followed by FaqI (Thermo Scientific, San Jose, CA, USA) and
BsrI (New England Biolabs, Ipswich, MA, USA) digestion, respectively.
The alas1smu350/smu350 PCR products were digested by FaqI into two
fragments of 57 bp and 85 bp, the alas1Δ2/Δ2 PCR products were digested
by BsrI into two fragments of 43 bp and 97 bp, whereas the WT PCR
products were resistant to the digestion. Primers for alas1 mutants
genotyping were as follow: 5'-GGACTCGGTCATGCACAAGAT-3' and
2
5'-GCTGGGTCACTGAAAACACCA-3'. gata1am651/m651 mutants were
genotyped by PCR followed by TaqI (New England Biolabs, Ipswich,
MA, USA) digestion. The WT PCR products were digested by TaqαI into
two fragments of 121bp and 123bp, whereas the gata1am651/m651 PCR
products were resistant to the digestion. Primers for gata1am651/m651
genotyping were as follow: 5'-GTGAGTATACACAATTACAC-3' and
5'-GGTTCAGAGAATACGCTCCT-3'. The digested products were
analyzed by high-resolution melting (HRM) or electrophoresis. Especially,
as the swim bladder was absent in alas1 mutants, alas1 mutants could be
directly distinguished from their siblings under stereomicroscope from 4
dpf onwards. As the gata1am651/m651 mutants were lack of erythrocytes,
gata1am651/m651 mutants could be directly distinguished from their siblings
under stereomicroscope from 24 hpf on"

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Download the product protocol from Thermo Fisher Scientific for FaqI (BsmFI) (2 U/µL) below.

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