TaaI (HpyCH4III) (10 U/µL)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Because of presence of another restriction site for TaaI (nucleotide 2643–2647, ACT/GT) rather than recognition target site (nucleotide 2710 2714, ACT/GT), straight application of TaaI on the first PCR products was inaccurate. For elimination of first restriction site another semi nested PCR was performed using P5 as a forward and P6 as a reverse primer (Table1). Subsequently amplified fragment (225bp) was digested with TaaI. Briefly the PCR product was first purified using PCR purification kit (MBST,Iran).	fter purification the 10 µl of each purified PCR product was digested with 2 µl TaaI(Fermenta,10U/ µl) in 2 µl 10× buffer Tango and 16 µl nuclease-free water for 16h at 65°C. As enzyme functional control, first PCR product (403bp) was digested with the restriction enzyme. In some cases, the nucleotide sequence of PCR product was determined through Kowsar Company (Iran).

Upstream tips
Because of presence of another restriction site for TaaI (nucleotide 2643–2647, ACT/GT) rather than recognition target site (nucleotide 2710 2714, ACT/GT), straight application of TaaI on the first PCR products was inaccurate. For elimination of first restriction site another semi nested PCR was performed using P5 as a forward and P6 as a reverse primer (Table1). Subsequently amplified fragment (225bp) was digested with TaaI. Briefly the PCR product was first purified using PCR purification kit (MBST,Iran).
Protocol tips
fter purification the 10 µl of each purified PCR product was digested with 2 µl TaaI(Fermenta,10U/ µl) in 2 µl 10× buffer Tango and 16 µl nuclease-free water for 16h at 65°C. As enzyme functional control, first PCR product (403bp) was digested with the restriction enzyme. In some cases, the nucleotide sequence of PCR product was determined through Kowsar Company (Iran).

Publication protocol

From all specimens, 34 numbers were selected in order to examine with PCR-RFLP method was described by Tiwari et al. (12).We decided to complete this method on first PCR products (403 bp). Because of presence of another restriction site for TaaI (nucleotide 2643–2647, ACT/GT) rather than recognition target site (nucleotide 2710 2714, ACT/GT), straight application of TaaI on the first PCR products was inaccurate. For elimination of first restriction site another semi nested PCR was performed using P5 as a forward and P6 as a reverse primer (Table1). Subsequently amplified fragment (225bp) was digested with TaaI. Briefly the PCR product was first purified using PCR purification kit (MBST,Iran). After purification the 10 µl of each purified PCR product was digested with 2 µl TaaI(Fermenta,10U/ µl) in 2 µl 10× buffer Tango and 16 µl nuclease-free water for 16h at 65°C. As enzyme functional control, first PCR product (403bp) was digested with the restriction enzyme. In some cases, the nucleotide sequence of PCR product was determined through Kowsar Company (Iran).

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 TaaI / HpyCH4III using TaaI (HpyCH4III) (10 U/µL) from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for TaaI (HpyCH4III) (10 U/µL) below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Restriction Enzymes TaaI / HpyCH4III using TaaI (HpyCH4III) (10 U/µL) 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.