mutagenesis - start to finish
Posted 06 July 2010 - 08:42 PM
1. extract RNA from tissue
2. convert to cDNA
3. clone into a vector (suggestions? using PCR sticky ends or Restriction Enzyme digestion)
4. transform into E.coli (DH5a)
5. screen w/ PCR and antibiotic/ blue/white screenin (dependant on vector used)
6. carry out site directed mutagenesis a la Ho et al 1989 (or the Altered sites mutagenesis kit from promega if i can convince my boss to buy it)
7.sequence positive clones
may seem like a stupis question but I'm able to express and purify my protein out of the E.coli aren't I?
Like Isaid - it's been a while and getting back on the bike is NOT as easy as I thought it would be
Posted 15 July 2010 - 08:04 PM
Posted 16 July 2010 - 04:36 PM
The attached paper worked well for me, with the addition of a ligase step after the PCR, but before the Dpn1 digestion.
I can recommend using Topo TA cloning kits which are very quick and work extremely well.