wrong cut size - (Apr/07/2008 )
When i cut pcDNA3.1+ with NheI/EcorI, I got a band the size was around 700bp. I can't figure out what's going on. Supposed it should 57bp.
-yqlu-
Are you sure that you have the right plasmid?
-zek-
QUOTE (zek @ Apr 7 2008, 10:30 AM)
Are you sure that you have the right plasmid?
I have checked with PstI, and I got the right fragments.
-yqlu-
EcoRI could have exhibited some star activity if the conditions allowed it..
-MolBioGirl-
What are your digestion conditions?
Maybe you can give some more info? Have you tried with another batch of enzyme or buffer?
-zek-
QUOTE (MolBioGirl @ Apr 7 2008, 02:50 PM)
EcoRI could have exhibited some star activity if the conditions allowed it..
good to know that, thank you.
-yqlu-
QUOTE (zek @ Apr 7 2008, 03:19 PM)
What are your digestion conditions?
Maybe you can give some more info? Have you tried with another batch of enzyme or buffer?
Maybe you can give some more info? Have you tried with another batch of enzyme or buffer?
DNA 3ul (1ug/ul)
EcorI 3ul
nheI 3ul
Buffer I 6ul
BSA 1ul
dH2O 44ul
37C O/N
-yqlu-
What brand of reagents are you using? If you're using NEB, they recommend buffer EcoRI (and BSA), since it minimizes star activity. Also, overnight is kind of long -- I would only run this for about 4 hours. That should solve it.
Good luck! 
-MolBioGirl-
QUOTE (MolBioGirl @ Apr 7 2008, 03:39 PM)
What brand of reagents are you using? If you're using NEB, they recommend buffer EcoRI (and BSA), since it minimizes star activity. Also, overnight is kind of long -- I would only run this for about 4 hours. That should solve it.
Good luck!
Good luck!
NEB, I don't know if i can use buffer ecori for nhei. Thanks again. and i like your signature.
-yqlu-
QUOTE (yqlu @ Apr 7 2008, 02:49 PM)
QUOTE (MolBioGirl @ Apr 7 2008, 03:39 PM)
What brand of reagents are you using? If you're using NEB, they recommend buffer EcoRI (and BSA), since it minimizes star activity. Also, overnight is kind of long -- I would only run this for about 4 hours. That should solve it.
Good luck!
Good luck!
NEB, I don't know if i can use buffer ecori for nhei. Thanks again. and i like your signature.
When in doubt, you can check the NEB double-digest recommendations here: http://www.neb.com/nebecomm/DoubleDigestCalculator.asp
(I use it every time I do a double digest)
Their website also has a lot of really good information in general about setting up the reactions. I consult it often.
Thanks! I'm glad you like the sig -- it's true!
-MolBioGirl-