FastDigest SspI

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Twenty microlitres of PCR amplification were digested in a 40 μL volume containing 1× FastDigest Green buffer supplemented with tracking dyes, 1 μL of 20 mg/mL acetylated BSA(Ambion) and 1 μL of FastDigest PstI, SspI, EcoRI and ClaI enzymes (Thermo Scientific). Reaction mixtures were incubated at 37 °C for 15 min then directly loaded onto the gels. At the same time, 10 μL of digested fragments from lpdA gene was examined by electrophoresis in 2% agarose gel, containing 1% agarose for routine use (Sigma) and 1% agarose low melting point.

Protocol tips

Twenty microlitres of PCR amplification were digested in a 40 μL volume containing 1× FastDigest Green buffer supplemented with tracking dyes, 1 μL of 20 mg/mL acetylated BSA(Ambion) and 1 μL of FastDigest PstI, SspI, EcoRI and ClaI enzymes (Thermo Scientific). Reaction mixtures were incubated at 37 °C for 15 min then directly loaded onto the gels. At the same time, 10 μL of digested fragments from lpdA gene was examined by electrophoresis in 2% agarose gel, containing 1% agarose for routine use (Sigma) and 1% agarose low melting point.

Publication protocol

lpdA gene sequences from Mmc strain GM12 (NCBI, nucleotide ID 256385136) and Mcc strain ATCC 27343 (NCBI, nucleotide ID 83319253:275328–277217) were aligned using the EMBOSS pairwise alignment algorithm.1 The primers LPD-C1-F and LPD-C1-R (Table 1) were selected based on the conserved region of the lpdA gene between the two Mycoplasma spp. PCR reactions were conducted in a DNA thermal cycler (GeneAmp 9700, Applied Biosystems). PCR products were purified and submitted to BMR Genomics2 for sequencing. The sequences of the lpdA gene from reference strains and isolates were compared with those of the GenBank database by using the BLASTN local alignment search tool.3 Enzymes and lengths of resulting restriction fragments were predicted with an on-line program.4 Twenty microlitres of PCR amplification were digested in a 40 μL volume containing 1× FastDigest Green buffer supplemented with tracking dyes, 1 μL of 20 mg/mL acetylated BSA(Ambion) and 1 μL of FastDigest PstI, SspI, EcoRI and ClaI enzymes (Thermo Scientific). Reaction mixtures were incubated at 37 °C for 15 min then directly loaded onto the gels. At the same time, 10 μL of digested fragments from lpdA gene was examined by electrophoresis in 2% agarose gel, containing 1% agarose for routine use (Sigma) and 1% agarose low melting point

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 SspI using FastDigest SspI from Thermo Fisher Scientific.

Manufacturer protocol

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

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Restriction Enzymes SspI using FastDigest SspI 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.