FastDigest BclI

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Generation of BclI-rings: Circularization of BclI-digested gDNA molecules into DNA rings was performed as was described by Rossetti et al. [13, 16]. An updated circularization reaction entails (i) digestion of 1 μg of gDNA with 10 U of Fast Digest BclI restriction enzyme (Thermo Scientific, Argentina) in a volume of 30 μL by 30 min at 37 °C, followed by heat inactivation 20 min at 80 °C and ethanol precipitation; (ii) fragment-ends’ self-ligation with 3 Weiss U/0.5-μg-gDNA of T4 DNA Ligase (Thermo Scientific, Argentina) in a volume of 200 μL (5 ng gDNA/μL) at 22 °C for 1 h, followed by ethanol precipitation and resuspension in 30 μL of distilled water (approximately 33 ng/μL).

Protocol tips

Generation of BclI-rings: Circularization of BclI-digested gDNA molecules into DNA rings was performed as was described by Rossetti et al. [13, 16]. An updated circularization reaction entails (i) digestion of 1 μg of gDNA with 10 U of Fast Digest BclI restriction enzyme (Thermo Scientific, Argentina) in a volume of 30 μL by 30 min at 37 °C, followed by heat inactivation 20 min at 80 °C and ethanol precipitation; (ii) fragment-ends’ self-ligation with 3 Weiss U/0.5-μg-gDNA of T4 DNA Ligase (Thermo Scientific, Argentina) in a volume of 200 μL (5 ng gDNA/μL) at 22 °C for 1 h, followed by ethanol precipitation and resuspension in 30 μL of distilled water (approximately 33 ng/μL).

Publication protocol

"Generation of BclI-rings: Circularization of BclI-digested gDNA molecules into DNA rings was performed as was described by Rossetti et al. [13, 16]. An updated circularization reaction entails (i) digestion of 1 μg of gDNA with 10 U of Fast Digest BclI restriction enzyme (Thermo Scientific, Argentina) in a volume of 30 μL by 30 min at 37 °C, followed by heat inactivation 20 min at 80 °C and ethanol precipitation; (ii) fragment-ends’ self-ligation with 3 Weiss U/0.5-μg-gDNA of T4 DNA Ligase (Thermo Scientific, Argentina) in a volume of 200 μL (5 ng gDNA/μL) at 22 °C for 1 h, followed by ethanol precipitation and resuspension in 30 μL of distilled water (approximately 33 ng/μL).

Inverse-PCR reactions were performed in separate reactions from 8 μL (roughly 260 ng) of BclI-circles for amplification of the F8-wild-type allele (primers 22B + ID) and the Inv22 allele (primers 22B + ED): yielding products of 2.5 and 2.6 kb, respectively, using 0.6 μM of each primer (Table 1), 1.5 U of Go Taq® DNA Polymerase (Promega) and standard PCR reagents in a total volume of 20 μL. Thermo-cycling conditions: 94 °C for 3 min (1 cycle), 94 °C 30 s; 52 °C 1 min and 72 °C 6 min (30 cycles) and 72 °C 6 min (1 cycle) on a thermal-cycler Life Express TC-96/G/H (Bioer). Inverse-PCR products were analyzed by ethidium bromide-stained 1% agarose gel electrophoresis.

Table 1 Oligonucleotide primer sequences, physical map locations, and references
Full size table
F8Int21[CA]n STR locus genotyping was performed using 2 µL of 10−6 or 10−7 serial dilution of each Wt- and Inv22-allele-specific iPCR product as a substrate of a nested-PCR amplification with primers F8Int21F and F8Int21R (Table 1). Alternatively, agarose gel blocks containing each allele-specific iPCR band (i.e., Wt: 2529 bp and Inv22: 2601 bp) were excised and suspended separately in 300 µL of distilled water overnight. F8Int21[CA]n STR nested-PCR substrate analyses using ten-fold serial dilutions ranging from 10−1 to 10−3 for each allele-specific iPCR product showed that 2 µL of the 10−3 dilution of each 300 µL preparation of Wt- and Inv22-allele were required for optimal STR amplification.

Also, the PacI restriction enzyme was used to generate PacI-rings, substantially as described above, over PacI digested long linear gDNA molecules of 28.5 kb. Inverse-PCR assays were performed separately from 8 μL of PacI-rings for amplification of the F8-Wt allele not involved in Inv22 (primers Int20PH + Int22PH, Table 1) yielding a product of 3.1 kb in the presence of 0.6 μM of each primer, 1.75 mM MgCl2, 0.75 U of Kapa Long-Range PCR System (BioSystems), and standard PCR reagents in a total volume of 20 μL. Thermo-cycling conditions were equal to those described for BclI-circles. Inv22 type 1 allele associates with an extremely long PacI-ring of 69.9 kb, thus hindering their formation and impairing their iPCR analysis.

For the third PI application over the PacI digested long linear gDNA molecules of 28.5 kb rings, iPCR assays were performed separately for amplification of the F8-wild-type allele (primers Ex20[A]wt + Int22PH) and the variant allele (primers Ex20[G]var + Int22PH) yielding products of 3.04 kb comprising either the c.6118T-wt or c.6118C-var allele (Table 1).

F8Int22[GT]n STR genotyping with primers F8Int22F and F8Int22R was performed from agarose gel blocks containing each allele-specific iPCR band (i.e., PacI Wt: 3,130 bp for Inv22 phasing; PacI Wt or Var allele-specific amplification for F8:c.6118 T > C: 3,035 bp) excised and suspended separately in 300 µL of distilled water overnight. F8Int22[GT]n STR nested-PCR substrate analyses using ten-fold serial dilutions ranging from 10−1 to 10−3 for each allele-specific iPCR product showed that 2 µL of the 10−1 dilution of each 300 µL preparation of Wt for Inv22 phasing, and Wt and Var c.6118 T > C alleles were required for optimal STR amplification."

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Restriction Enzymes BclI using FastDigest BclI from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for FastDigest BclI below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Restriction Enzymes BclI using FastDigest BclI from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.