New England Biolabs, R0745S, PaqCI®

PaqCI has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10211228. Related Categories Restriction Endonucleases P R Applications High-throughput cloning and automation solutions,, NEBridge® Golden Gate Assembly Specification Unit Definition One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 μl. Reaction Conditions 1X rCutSmart™ Buffer Supplement with PaqCI Activator Incubate at 37°C rCutSmart™ Buffer 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Activity in NEBuffers NEBuffer™ r1.1: 10% NEBuffer™ r2.1: 100% NEBuffer™ r3.1: 10% rCutSmart™ Buffer: 100% Diluent Compatibility Diluent B Storage Buffer 10 mM Tris-HCl 300 mM NaCl 1 mM DTT 500 µg/ml Recombinant Albumin 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 20 minutes Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Not Sensitive CpG Methylation: Impaired by Overlapping FAQ Q: Which restriction enzymes are used in Golden Gate Assembly? A: Golden Gate Assembly is a one-tube efficient cloning method based on Type IIS restriction enzymes that cleave outside their recognition sites and leave 3 or 4-base overhangs. BsaI is the most commonly used Type IIS enzyme for Golden Gate Assembly. NEB also offers an engineered high-fidelity version of this enzyme, BsaI-HF®v2, which has the added benefit of being Time-Saver™ qualified (can digest DNA in 5-15 mins or overnight without degradation to DNA) and exhibits dramatically reduced star activity. Other Type IIS enzymes used in Golden Gate Assembly include BsmBI-v2/Esp3I, BbsI/BbsI-HF, PaqCI (AarI isoschizomer with 7 bp recognition sequence), and SapI/BspQI/BspQI-HF (with 7bp recognition sequence and 3bp overhangs). Q: Which restriction enzymes are used in GoldenBraid Assembly? A: BsaI is one Type IIS enzyme used in this method. NEB also offers an engineered high-fidelity version of this enzyme, BsaI-HF®v2, which has the added benefit of being Time-Saver™ qualified (can digest DNA in 5-15 mins and can be digested safely overnight) and exhibits reduced star activity. BsaI-HFv2 works in CutSmart® Buffer, as does T4 DNA Ligase (also part of the GoldenBraid workflow) which is 100% functionally active in this buffer, when the reaction is supplemented with 1 mM ATP. Other Type IIS enzymes used in GoldenBraid include BsmBI-v2/Esp3I, BtgZI, BbsI/BbsI-HF and PaqCI (AarI isoschizomer with 7 bp recognition sequence). Reference: GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology Q: When is star activity a concern? A: Star activity is of concern if extra banding can cause misinterpretation of results in genotyping and mutational analysis procedures. Experimental design can promote star activity. Small reaction volumes are more likely to contain glycerol concentrations of 5% or greater, a condition known to increase star activity. A 5% glycerol concentration occurs when setting up a double digest in a 20 µl reaction using 1 µl of each enzyme. Overnight digests are more likely to generate star activity. For tips on avoiding star activity, please click here. Q: Why is PaqCI not cutting DNA to completion? A: PaqCI is an enzyme that requires multiple recognition sites to achieve complete digestion. NEB has therefore included “PaqCI Activator” which can be added to the reaction with 1:1 ratio (i.e. 1 ul enzyme to 1 ul PaqCI Activator). Please note that for Golden Gate Assembly reactions, the amount of PaqCI Activator varies depending on how many fragments are being assembled. Please visit Usage Guidelines for Golden Gate Assembly with PaqCI for additional information. Q: Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers? A: NEB is excited to announce that we have switched BSA-containing reaction buffers (NEBuffer 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We are also in the process of moving all restriction enzyme formulations to contain rAlbumin. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price. Q: For Golden Gate Assembly, how many base pairs should my amplicon inserts have flanking the PaqCI restriction site? A: Amplicon inserts must posses 5' flanking bases and PaqCI restriction sites at both ends of the amplicon in the proper orientation. We recommend adding 6 flanking bases at the 5' ends of the primers for optimal PaqCI binding, cleavage, and overall Golden Gate Assembly efficiency. This 6 base pair recommendation supersedes other known end length preferences for the enzyme due to Golden Gate Assembly protocols requiring maximum enzyme activity for efficient assembly.