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Restriction Endonuclease Buffers >
BSA > FAQ | | BSA FAQ
See the Buffers FAQ also.
Q1: What is the source of your purified BSA?
Q2: What is the buffer composition of your purified BSA?
Q3: Will the buffer from the BSA inhibit my reaction?
Q4: What quality controls are performed on your purified BSA?
Q5: Why do I need to add BSA to my restriction digest?
Q6: When setting up a restriction digest, if BSA is not listed as a reaction component, can it have a negative effect if added?
Q7: Is the BSA in its native form?
Q8: Can I use your BSA as a molecular weight marker?
Q9: Can I use your BSA as a concentration standard?
Q10: Is there a difference between purified BSA and the acetylated BSA you sold previously?
Q1: What is the source of your purified BSA?
A1: The source of NEB purified BSA is a molecular biology grade, protease free, fraction V starting material. This material is then processed to remove nucleases.
Q2: What is the buffer composition of your purified BSA?
A2: The buffer composition is 20mM KPO4 (pH 7.0 at 25°C), 50 mM NaCl, 0.1 mM EDTA and 5% glycerol.
Q3: Will the buffer from the BSA inhibit my reaction?
A3: The BSA should be diluted 1:100 for standard reactions. At this level the buffer from the BSA should have little effect.
Q4: What quality controls are performed on your purified BSA?
A4: A 16 hour incubation is performed with ΦX174 DNA- HaeIII Digest (NEB# N3026). This assay checks for endonuclease, exonuclease and binding protein contamination.
A 16 hour incubation is performed with ΦX174 RFI DNA (NEB# N3021). This is a sensitive assay for nonspecific endonuclease.
A 1 hour incubation with a RNA ladder is performed. This assay checks for RNAse.
Q5: Why do I need to add BSA to my restriction digest?
A5: Adding BSA to a reaction lessens enzyme loss on tube and pipette tip surfaces. BSA stabilizes enzymes in reaction. The stabilizing effects are most pronounced in overnight reactions (Robinson D. unpublished observation).
Q6: When setting up a restriction digest, if BSA is not listed as a reaction component, can it have a negative effect if added?
A6: Restriction enzymes that do not require BSA for optimal activity are not adversely affected if BSA is present in the reaction.
Q7: Is the BSA in its native form?
A7: The BSA is heat treated to inactivate nucleases during the purification process. Much of the BSA is denatured by this heat step.
Q8: Can I use your BSA as a molecular weight marker?
A8: Purified BSA (NEB# B9001) is not designed to be used as a molecular weight marker. Our Protein Marker , Broad range (NEB# P7702) uses BSA as one of its marker bands. This BSA used in the protein marker is selected for size and the absence of breakdown products. The molecular weight of BSA is 66,409 daltons. Purified BSA (NEB# B9001) run on SDS PAGE will give a major band at this size. We do not guarantee that it will give a single sharp band when used in this manner.
Q9: Can I use your BSA as a concentration standard?
A9: Purified BSA (NEB# B9001) is not designed to be used as a concentration standard. Due to the scale of purification and the nature of the filtration step used in processing; the 10 mg/ml concentration is a rough estimate. We do not suggest that this product be used as a concentration standard.
Q10: Is there a difference between purified BSA and the acetylated BSA you sold previously?
A10: Yes, a heat step was substituted for the chemical acetylation used to inactivate nucleases in earlier preparations. The new process is more effective and removes the possibility of acetyl transfer between proteins that could happen at elevated reaction temperatures.
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