TITLE: Gibson protocol from RJ communications
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Gibson protocol </div></div>

[STEPS]
?. [Protocol from RJ and SH]
Perform vector and insert amplification using Q5 PCR
?. [Protocol from RJ and SH]
Determine which reactions had successful amplification via agarose gel electrophoresis, use the reaction with a strong/bright band and the lowest amount of starting template for the following steps.
?. [Protocol from RJ and SH]
Common: Treat vector with DpnI if final selection method would also select for vector template.
?. [Protocol from RJ and SH]
**Do not PCR purify before treating with DpnI unless you need to do gel extraction. DpnI has activity in the Q5 buffer.
?. [Protocol from RJ and SH]
Rare: treat inserts with DpnI if, for some reason, the template would be selectable with the final selection method.
?. [Protocol from RJ and SH]
PCR purify template and inserts using Qiagen kit or gel extract
?. [Protocol from RJ and SH]
Determine the DNA concentration of each piece using a fluorimeter (nanodrop can be less reliable according to Dr. Fozo).
?. [Protocol from RJ and SH]
Calculate concentrations and volumes you will need to performGibsonassembly reaction before setting up the reaction(s).This is especially important if you are doing multiple reactions.If you do not have enough of each piece, repeat the previous steps until you do
?. [Protocol from RJ and SH]
Perform Gibson assembly reaction
?. [Protocol from RJ and SH]
Assemble in Eppendorf tube10µL 2X Gibson assembly Master MixDNA as needed 50 to 100 ng vector, 5:1 or 3:1 molar ratio insert:vector for each insert. Use Nebiocalculator.neb.comThe total volume of vectors and insert should not exceed 10µLddH2O to 20µL Include a no-insert control only if required 50 to 100 ng vector, 5:1 or 3:1 molar ratio insert:vector for each insert. Use Nebiocalculator.neb.comddH2O to 20µL Include a no-insert control only if required
?. [Protocol from RJ and SH]
Transform 2µL Gibson assembly into TOP10 cells (or another appropriate strain)using electroporation procedure. Rescue with 500µL media (SOC, etc.), plate 100-125µL rescue on 4-5selective plates.
?. [Protocol from RJ and SH]
Highly suggested: Perform PCR check of Gibson assembly prior to transformation to see if Gibson was successful.
?. [Protocol from RJ and SH]
Incubate 1hr at 48oC in heat block (can go longer than 1hr)
?. [Protocol from RJ and SH]
Streak isolated colonies, 4-8 for each Gibson, for isolation on appropriate selective media.
?. [Protocol from RJ and SH]
Screen colonies after isolation streaking using colony PCR for successful Gibson (same PCR as step 8). Use empty vector as control for amplification.
?. [Protocol from RJ and SH]
Quickly determine presumptively positive isolated colonies from colony PCR, perform steps to prepare -80oC temporarily frozen stocks.
?. [Protocol from RJ and SH]
At the same time as step 12, isolate plasmid and send out for sequencing if required to confirm no unwanted mutations have occurred and that your colony is fully isolated.
?. [Protocol from RJ and SH]
If sequencing looks good, move from temporary -80oC to long-term -80oC storage by communicating with Dr. Fozo.