Year of the elusive plasmid

 For the last year, starting in July 2023, we have been attempting to assemble and verify a plasmid to use in the transformation of rice plants.  Two scientists from Johns Hopkins University visited us last July.  They brought the insert containing the gene to express GRFT.  Having no previous experience with plasmid assembly, this task involves a learning curve for me.  There have been multiple fails which are among other things, teachers.   We try not to repeat the same mistakes and like to think we are making progress.     

It's an interesting summer that hasn't left much time for posting on this blog.  We're still alive and kickin'.

  

DNA plasmid under construction

Project GRFT update.  Sreenivas Eadara and the Project GRFT DNA crew in Baltimore are making good progress with the plasmid construct assembly.  

Beautiful bands:

important considerations when preparing projectiles

 Basically when coating particles we need to pay careful attention to:

1. pH: This is most important!
2. Timing of incubation
3. Temperature of incubation
4. Salt concentration
5. Size of particles
6. Freshness of Spermidine (0.1M)
7. 2.5 M CaCl2 is filter sterilized and aliquots prepared ready to use.
8. Purity and concentration of DNA
9. Keeping particles suspended in the buffer solution while adding the spermidine and CaCL2
10. We have seen problems with particles sticking to the 1.5 Ml centrifuge tubes. DNA Lo-Bind tubes should work better.