Posted 06 August 2004 - 06:48 AM
Posted 06 August 2004 - 05:32 PM
Posted 07 August 2004 - 10:17 AM
Posted 12 August 2004 - 05:05 AM
If in doubt, you could digest with your first enzyme, clean up and/or adjust the buffer and digest with your second enzyme.
How do you do the transformation? Electroporation? Do you re-use the cuvettes?
Do your agar plates contain the appropriate antibiotics? Perhaps they are too old?
Posted 12 August 2004 - 08:29 AM
You may also get very high density with electroporation ausing an uncut vector if you plate a lot of the transformation.
Make new plates, make sure your bugs are OK and do it again.
Posted 12 August 2004 - 11:13 AM
For cutting with EcoRI/XhoI i also cut another bector having my insert with the same enzymes and it worked fine so I assume that the empty pFLAG vector too would be cutting fine with the two enzymes.
Thanks for your help!
Posted 12 August 2004 - 04:38 PM
molbiouser, on Aug 12 2004, 12:13 PM, said:
It's also not endogenous resistance in the bugs since you can get vector back.
Now for the lame questions...How much vector did you cut with how much enzyme in what volume? Did you see a nice single vector band in the gel purification? How much vector did you use in the ligation? Did you accidentally take vector from the uncut vector tube instead of the cut. This has been the cause of much head scratching on an occasion or two with people in various labs I've been at.
Posted 13 August 2004 - 02:13 AM
1. When you cut the band out of the gel, you are also cutting undigested vector (there's ALWAYS some undigested material). the solution to this is to run the undigested, original plasmid next to the digested vector and make sure you are cutting at an entirely different site (if possible). Also, run a high percentage gel, >2%
2. Antibiotic; I've fucked up this bit big time and took me a year (!) to realise. For ampicillin (or carbenicillin), make a stock of 100mg/ml (and use at 100um/ml) by weighting out 1 gram and resuspending it in 10 ml. The mistake I was making was that I was calculating the MW of amp, ending up with 100mM instead of 100mg/ml...
Posted 13 August 2004 - 08:35 AM
Also, higher percent gels are usually done to distinguish smaller particles, lower percent to separate vector sized ones...at least in my experience.