2 replies to this topic

[kb_012]

Hi, I've recently come from bacteria into mammal work and have been out of the field for ~10mths being a FT mummy. I've been thrown in the deep end a bit and am not sure I'm going about this the right way. as the lab I'm in doesn't have anyone with any particular expertise in this field I'm at a loss. i need to perform single aa mutagenesis on a kinase however the lab doesn't have any starting point stock for me. so here's my plan - suggestions very welcome.
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

[jangajarn]

seems good to me.. convince your boss with reg to Quik change mutagenesis kit from stratagene... you wont regret. for step 4, try whatever suits your final objective best. i.e. if you are going to express your mutagenized construct in cells, clone your gene into that plasmid, to avoid a subcloning step.

[bob1]

Quikchange works well, but you definitely don't need to buy the kits... all you need are SDM primers (which you can design for free on the Promega site), a proofreading polymerase and a T4 ligase.

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.

Attached Files

•  SDM_primers.pdf   174.06K   85 downloads