Registry of standard biological parts/PCR-based assembly/Protocol

=Plan Construction= As an example we will be building:
 * 1) J04500 + I13501 = J04450 (RFP expression device)
 * 2) *Promoter/RBS + FP

Possibly try this later:
 * 1) R0010 + I13504 = J04430 (GFP expression device)
 * 2) *Promoter + RBS/FP

=Primer Design=

External Primers

 * 1) Design 20bp primers from the ends of the finished assembly.
 * 2) Add 15bp overhangs based on digesting 1AK3 with E/P using this primer design tool for primers for the In-Fusion cloning method

Fusion primers
=PCR Reactions=

PCR conditions

 * Used the same conditions for J04500 and I13501
 * 1) Initial Denaturation – 98C for 30s
 * 2) Denaturation – 98C for 5s
 * 3) Annealing – 65C for 15s @ Tm+3C
 * 4) Extension – 72C 13s
 * 5) repeat to (2) 20 times.
 * 6) Final extension – 72C for 5-10min
 * 7) 4C indef

PCR Reaction B

 * Going to try this 2 ways:
 * Dilute the PCR produces of PCR A 1/1000 (add 1uL of each as the template) and then add them together with external primers
 * Do extension with no primers and add the undiluted PCRA as primers (e.g. 5uL of each one with no template added).

PCR mix #2
=References= Sauer:Stitching Genes by PCR