"

From 2014.igem.org

---

Revision as of 22:43, 1 July 2014

Core primers

Masp1 Overlap 18 long, 54 Tm, 56% GC Calculated Tm of core “primer”: 62 C, less than 20 long so annealing should be roughly equivalent to Tm Actual Protocol MaSP 1 Core primers (60.1 by thermosci) Mix and centrifuge components prior to use Assembling on ice, add Phusion Polymerase last, transfer to Thermocycler heated to 98 C 12.8uL Nuclease-free water 4 uL 5X Phusion HF or GC buffer 0.4 uL 10 mM dNTPs 1uL 10 uM Forward Primer 1uL 10uM Reverse Primer 0.6 uL DMSO, (optional, if not used add 0.6 uL water) 0.2 uL Phusion DNA Polymerase

Step	Temperature	Time
Initial Denaturation	98 C	30 seconds
35 Cycles Denaturation Annealing Elongation	98 60 72	10 sec 30 sec 10 sec
Final Extension	72	10 minutes
Hold	4-10