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Restriction enzyme digestion

Restriction enzyme digestion - Transfection Forum

Restriction enzyme digestion - Transfection Forum. Cell transfection discussion including stable cells transfection, transient transfection. Troubleshooting and posting of discussions on lipofection, and reagents: Fugene, lipofectamine, and others.


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Old 03-17-2009, 03:52 PM
Pipette Filler
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Default Restriction enzyme digestion



We have digest a plasmid that comprises vector pcDNA 6.2/EmGFP TOPO and 1,6 kb of insert. We have use EcoRI and BamHI we expect to see three bands, but we obtained negative results.
This is the protocol used:
1,5 l specific buffer
0,5 l enzyme (5 U/reaction)
1 g DNA
Vol tot 15 l H2O DEPC
Incubation 2 h at 37C
HELP PLEAS
HELP PLEAS
We need to get any indication to obtain positive results.
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Old 01-23-2010, 01:26 PM
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Default Re: Restriction enzyme digestion

may be your enzymes r not working try fast digest enzyme fermentas. check insert with pcr using specific primers
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Old 01-24-2010, 09:42 PM
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Default Re: Restriction enzyme digestion

negative results? how many bands do you see?

where do the enzymes cut? insert and/or vector?

have this insert been analysed before, so that you know its correct?
otherwise my guess is that your insert is not what you think..

If the digestion is the problem, get new enzymes.
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digestion , enzyme , restriction


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