Published: March 21st, 2020 Last Modified: April 16th, 2020
I’ve got the Mashup RT plasmid (and everything else on the list) to send out to academics/health care providers running out of commercial enzymes and kits, get in touch through the contact form.
!!! Most up to date Mashup-RT Purification Protocol !!!
PDF, DOCX
V1.2 Update:
– DTT should be added to final concentrated protein at a concentration of 1mM. My mistake, please accept my apologies. Dropped by accident, thank you kind reader for informing me.
V1.1 Update:
– Added section for FPLC users
– Added representative gels of purification
– Bumped up imidizole in Lysis Buffer from 20 to 40mM
– Bumped up imidizole in Wash buffer from 50 to 80mM
FAQ:
- Yes, Mashup is thermostable, active up to 70C, albeit at a reduced activity. Typical reaction temperatures are 50-55C
- The yield from 1 liter of culture is 250000 to 500000 reactions
- My concentrated stock is approximately 0.5 mg/mL, going higher than this will trigger precipitation
- Quantify Mashup with the BCA assay, Bradford does NOT work
Please leave corrections/additions in comments, thank you!
Great job !
I will try this.
Thanks
I have treid many mutants to impove the thermostablity of the MLV, but failed up to 65C. Only one mutant performed ~20% activity after 15min of treatment at 65C. So any one could test the thermostablity of Mashup RT at 70C? If so, it is really usefull.
Thanks!
Mashup is active up to 70C, albeit at a reduced activity.