Author: vrinaolo

Tested: NEB® 5-alpha Competent E. coli (High Efficiency) cells

Summary: cells perform 100x worse than advertised especially on the higher range of DNA

Summary: use a viscous polyvinylpyrrolidone (PVP) solution as a sealant

Summary: Bst 3.0 is 3-24x higher yielding than homemade MMLV

kg/mol x pmol = ng

μM x μL = pmol

μM x kg/mol = ng/μL

I needed to prepare a plasmid library and was curious which had the best yield

Bottom line: they are about the same for two component assembly

• By gel both rxns are about 25% efficiency
  • Both rxns have about 3ng/uL backbone/vector and yield about 0.75ng/uL of plasmid
• They gave a similar number of colonies when transformed

Instructions

Use 0.2x (final) SYBR green in your qPCR mix. 

Notes

• You can do everything else as usual (PCR rxn setup and cycling parameters)
• SYBR green typically comes as a 10,000x stock in DMSO. You can dilute 1uL of 10,000x in 625uL of water and add 0.25uL of this dilution per 20uL qPCR rxn.
• I have done dozens of qPCR rxns with Taq, Phusion, AccuPrime and 0.2X SYBR and it has always worked.