BsrGI-HF®

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The PCR product was digested with BsrGI-HF and SalI-HF enzymes (NewEngland BioLabs), and the resulting 2,042-bp digested product (insert) was purified using agarose gel electrophoresis. The peGFP-N1 vector carrying the HA-mCherry-hSTIM1 was also digested with BsrGI-HF and SalI-HF enzymes, and the 4,780-bp vector backbone was purified by agarose gel electrophoresis.

Protocol tips
The PCR product was digested with BsrGI-HF and SalI-HF enzymes (NewEngland BioLabs), and the resulting 2,042-bp digested product (insert) was purified using agarose gel electrophoresis. The peGFP-N1 vector carrying the HA-mCherry-hSTIM1 was also digested with BsrGI-HF and SalI-HF enzymes, and the 4,780-bp vector backbone was purified by agarose gel electrophoresis.

Publication protocol

"DNA Constructs. YFP- and CFP-tagged STIM1/STIM2 constructs were obtained
from Tobias Meyer, Stanford University. YFP-STIM2ΔK5 was created as described previously (19). CFP-STIM2ΔSOAR was generated using the method
described earlier for YFP-STIM2ΔSOAR (19). mCherry-STIM2 was created
using the YFP-STIM2 as a template and replacing the YFP tag with mCherry.
The peGFP-N1 vector carrying the HA-mCherry-hSTIM1 was used as PCR
template to construct hSTIM1-ΔK with hSTIM2 K-rich domain (mCherrySTIM1-S2K). The 5′-GGC GAG GGC GAG GGC-3′ forward and 5′-TAT AGT
CGA CTC ACT TAG ATT TCT TCT TAA AAA GGC TTT TGA TTT TTG ATG GCT
TTT TGC TTT TGC CTG GGC TGG AGT CTG TTT C-3′ reverse primers were used
to generate a 2,647-bp PCR product. The forward primer annealed 105 nucleotides downstream of the start of the mCherry ORF. At 581 bp downstream of the primer annealing site, the mCherry contained an intrinsic BsrGI
restriction site (just before the end of the mCherry ORF). The reverse primer
was designed to anneal to the STIM1 region encoding for 664 to 670 residues (just upstream of the polybasic domain) and contained an overhang
encoding the STIM2-polybasic domain KSKKPSKIKSLFKKKSK, followed by a
stop codon and SalI restriction site.
The PCR product was digested with BsrGI-HF and SalI-HF enzymes (New
England BioLabs), and the resulting 2,042-bp digested product (insert) was
purified using agarose gel electrophoresis. The peGFP-N1 vector carrying the
HA-mCherry-hSTIM1 was also digested with BsrGI-HF and SalI-HF enzymes,
and the 4,780-bp vector backbone was purified by agarose gel electrophoresis. The insert and the vector backbone were ligated overnight at 16 °C
using T4 DNA ligase (New England BioLabs). The sequence of the final
construct was verified by Microsynth, Balgach, Switzerland. Orai1-CFP, YFPTubby, and rapamycin-inducible 5′ phosphatase were obtained from Tamas
Balla, Eunice Kennedy Shriver National Institute of Child Health and Human
Development. mCherry-STIM1 and mCherry-STIM1ΔK were provided by
Richard Lewis, Stanford University, and Myc-tagged STIM1 was provided by
Shmuel Muallem, National Institute of Dental and Craniofacial Research. The
mCherry-ER3 (ER marker), Myc- and GCaMP6f-tagged Orai1, and NFAT1-GFP
constructs were obtained from Addgene"

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 BsrGI / Bsp1407I using BsrGI-HF® from New England BioLabs.

Manufacturer protocol

Download the product protocol from New England BioLabs for BsrGI-HF® below.

Videos

Check out videos that might be relevant for performing Restriction Enzymes BsrGI / Bsp1407I using BsrGI-HF® from New England BioLabs. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.