Restriction Enzyme Digestion Troubleshooting Guide
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Restriction Enzyme Digestion Troubleshooting Guide
A nice troubleshooting guide and FAQs about Restriction Endonucleases from Fermentas.
Content:
Problems:
Undigested or incompletely digested DNA
Additional bands atypical for the banding pattern of a particular substrate
Diffused DNA zones - gel shift
Low efficiency of fragment ligation
Possible Reasons and Solutions:
1. Undigested or incompletely digested DNA
Nature of DNA.
Quality of DNA.
Recognition sequences.
Enzyme sensitivity to methylation.
Improper reaction conditions.
Improper dilution or addition of enzyme.
High glycerol concentration.
Partially or completely inactivated enzyme.
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Last update 28-Aug-2007, Rating Poor of 4 votes.
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it may the buffer problem or the prepation of plasmid minipreps, or the enzymes . so try with buffers and eznmes by keepnig them at 65C for 20 min or some times the running buffer it self has probelm
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Your plasmid DNA could either be incompletely digested or there might be undigested contaminating DNA in your plasmid DNA. Try performing the T4 ligase test for background check or phenol-extract your plasmid DNA after performing a miniprep
Rating: Very Good
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I am having problem with Restriction digestion of plasmid DNA with Xba 1 and BamH1. I am getting smear.
plz. help
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hello...i'm student from ukm...i just want to ask why when i do digestion...then run electrophoresis iget 3 band...i dont understand why?
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Related resource
Double restriction digestion of DNA
Rsa digestion.
Labelling of oligos: an interesting phenomenon
Non-radioactive labelling of DNA
High temp. restriction enzyme digests
RANDOM primer labelling efficiency
REBASER: The Restriction Enzyme Database
Integrated Enzyme Database (IntEnz)
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