TITLE: Construction of Mutant Library
AUTHORS: Shuning  Guo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to construct mutant library of target gene with high efficiency and low false positives/negatives rate after subsequent functional screening.</div></div>

[STEPS]
?. [Error-prone PCR]
Add the following reagent to a PCR tube (50μl) (Random Mutagenesis Kit by Solarbio).Template(10μl)Depends on the concentrationForward Primer (10 μM)1μlReverse Primer (10 μM)1μlMut Enhencer3μl2 x Mut Random System25μlddH2OAdd to 50μl
Template(10μl)Depends on the concentrationForward Primer (10 μM)1μlReverse Primer (10 μM)1μlMut Enhencer3μl2 x Mut Random System25μlddH2OAdd to 50μl
?. [Error-prone PCR]
Program the thermocycler as follows:TemperatureTime95℃2min94℃30 sTm-3~5℃1min72℃1kb/min72℃7 min16℃∞
TemperatureTime95℃2min94℃30 sTm-3~5℃1min72℃1kb/min72℃7 min16℃∞
?. [Error-prone PCR]
Use the palm centrifuge to mix the solution in PCR tube.
?. [Error-prone PCR]
Put the PCR tube into the thermocycler and Run the program.
?. [Error-prone PCR]
Using agarose gel electrophoresis to confirm if correct construct was present.
?. [PCR product purification]
PCR product purified by E.Z.N.A.® Cycle Pure Kit.
?. [MEGAWHOP PCR]
Add the following reagent to a PCR tube (50μl).Plasmid templete (50μl)Depends on the concentrationPurified Production of error-prone PCR (mega primer) (500μl)Depends on the concentration2×High Fidelity Master Mix (Enzyme)25 μlddH2OAdd to 50μl
Plasmid templete (50μl)Depends on the concentrationPurified Production of error-prone PCR (mega primer) (500μl)Depends on the concentration2×High Fidelity Master Mix (Enzyme)25 μlddH2OAdd to 50μl
?. [PCR product purification]
Test the concentration and purity of DNA using NanoDrop.
?. [MEGAWHOP PCR]
Program the thermocycler as follows:TemperatureTime95℃5min95℃30sDepends on the Tm30s72℃2kb/min72℃7 min16℃∞
TemperatureTime95℃5min95℃30sDepends on the Tm30s72℃2kb/min72℃7 min16℃∞
?. [PCR product purification]
PCR product purified by E.Z.N.A.® Cycle Pure Kit.
?. [PCR product purification]
Test the concentration and purity of DNA using NanoDrop.
?. [DpnI digestion]
Add the following reagents to a PCR tube (e.g. 20μl).DpnI (NEB) (20,000units/ml)Depends on the quality of DNA (20units DpnI degests 1μg DNA)10xCutsmart2μlPurified Production of MEGAWHOP PCR Moderate (e.g.400 ng)ddH2OAdd to 20μl
DpnI (NEB) (20,000units/ml)Depends on the quality of DNA (20units DpnI degests 1μg DNA)10xCutsmart2μlPurified Production of MEGAWHOP PCR Moderate (e.g.400 ng)ddH2OAdd to 20μl
?. [DpnI digestion]
Incubate at 37°C for 2 hours and heat Inactivation 80°C for 20 min.
?. [Digestion product purification]
Digestion product purified by E.Z.N.A.® Cycle Pure Kit.
?. [Digestion product purification]
Test the concentration and purity of DNA using NanoDrop.
?. [Nick ligation (T4 ligase)]
Add the following reagents to a PCR tube (e.g. 20μl)T4 DNA ligase (Thermo Fisher) (Weiss U)1UPurified Production of DpnI digestion50ng10X T4 DNA Ligase Buffer (Thermo Fisher)2μlddH2OAdd to 20μl
T4 DNA ligase (Thermo Fisher) (Weiss U)1UPurified Production of DpnI digestion50ng10X T4 DNA Ligase Buffer (Thermo Fisher)2μlddH2OAdd to 20μl
?. [Transformation]
Transform the nick ligation product into competent cells.
?. [MEGAWHOP PCR]
Use the palm centrifuge to mix the solution in PCR tube.
?. [MEGAWHOP PCR]
Put the PCR tube into the thermocycler and Run the program.
?. [MEGAWHOP PCR]
Using agarose gel electrophoresis to confirm if correct construct was present.
?. [DpnI digestion]
Use the palm centrifuge to mix the solution in PCR tube.
?. [Nick ligation (T4 ligase)]
Use the palm centrifuge to mix the solution in PCR tube.
?. [Nick ligation (T4 ligase)]
Incubate the reaction at 16℃ overnight.